MINISTRY OF HEALTH OF UKRAINE M.GORKY DONETSK NATIONAL MEDICAL UNIVERSITY

BIOCHEMISTRY

IN SCHEMES

PART 1

Donetsk2012

UDC 6612.015+577.1 (075.8)

Reviewed by:• Head of Histology, Cytology and Embryology Department of M.Gorky DonNMU,

prof. Eduard F. Barinov, PhD, MD

• Head of Molecular Genetic Research Laboratory of Central Scientific Research Laboratory of M.Gorky DonNMU, prof. Sergey V. Zyablitsev, PhD, MD.

• Head of Foreign and Latin Languages Department of M.Gorky DonNMU,

• Andrey A. Puzik, PhD.

Authors: Zoya M. Skorobogatova, Yuliya D. Tursunova, Igor I. Zinkovych,Anatoliy G. Matviyenko, Irina V. Nizhenkovska, Evgeniy V.Khomutov,Olga P. Shatova, Maryna A. Stashkevych

Editor of the English version: senior lecturer of World Literature and Classical Philology Department of Donetsk National University Olga V. Matviyenko, PhD

The present manual is recommended for publication by the Academic Council of M.Gorky DonetskNational Medical University (minutes № 7 dated 26.10.2012).

The manual is recommended for Medical University students learning biochemistry.

PREFACE

It is well-known that modern achievements in medical sciences are tightly associated with such basic discipline as biological chemistry. Biological chemistry, or biochemistry, studies the chemical composition and the chemical conversions taking place in living cells. New data concerning pathogenesis of various diseases as well as new criteria for their diagnostics and prognosis were revealed by means of biochemical methods of investigation. Due to the knowledge of biochemical processes occuring in human beings it is possible to synthesize new drugs for prophylaxis and treatment of diseases. All the above proves the importance of biochemistry as “the chemistry of life” for knowledge of maintenance of health.

Biochemistry is considered to be a difficult educational discipline. Moreover a great information content in biochemistry has been accumulated nowadays. For the most of students it is not simple to study and to analyze it. That is why the handbook “Biochemistry in Schemes” was prepared to represent the main biochemical pathways, their regulation and their possible disorders both in laconic graphic manner and in chemical formulae. The schemes and the pictures are accompanied by brief commentaries.

We suppose that the handbook “Biochemistry in Schemes” will facilitate to study the fundamental biochemical manuals and the special biochemical literature. According our point of view such all-round approach will help the students not only to understand and to study the subject, but also quickly recollect the main biochemical principles. The handbook may be adapted to learning another medical disciplines.

We hope that the handbook “Biochemistry in Schemes” will be interesting and useful for medical students.

The authors

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ENZYMES“Medicine speaks the language of enzymology” (A.Brownstein).

Enzymes are biological catalysts of protein nature that accelerate chemical reactions in the cell.Enzymes differ from other catalysts in three unique features:

high efficiency; high specificity; their ability to be regulated.

Evidence of enzymes protein nature:denaturation under the action of chemical

and physical factors;hydrolysis to amino acids;amphoterism;electrophoretic mobility;inability to diffuse through

the semipermeable membrane;sedimentation when salting-out;high molecular weight;high specificity;direct evidence – synthesis of enzymatic proteins.

Class1. Oxidoreductases

2. Transferases

3. Hydrolases

4. Lyases

5. Isomerases

6. Lygases(synthetases)

Type of reaction catalyzedOxidation –reduction reactions

Аred + Вox Аox + Вred

Transfer of atom groups from donor to acceptor moleculeА-Х + В А + В-Х

Bonds hydrolysisА-В + Н2О А-Н + В-ОН

Cleavage of bonds between atoms С, О, N, S in non-hydrolytic and non-redox way

А(Х)-В(Y) AX + BYIsomers interconvertion

А isoАFormation of bonds in the reaction of two molecules combination(with the use of ATP energy)

А + В А-ВATP ADP

Enzymes ClassificationIt is based on the type of the catalyzed reaction.

The primary structure of the first synthesized protein – enzyme ribonuclease (1969)

4

Enzymes Structure

•Enzymes are divided into two groups: simple and complex, or conjugated, enzymes.Simple enzymes are polypeptide chains that are broken down to amino acids by hydrolysis (for example, the enzymes of digestive tract: pepsin, lipase, amylase).Complex enzymes contain the non-protein part (cofactor) as well as the protein (apoenzyme) compound. If cofactor is tightly bound to the protein compound and it may not be detached during the enzyme separation it is named prosthetic group, for example, metal ions. The whole conjugated enzyme is named holoenzyme. If cofactor easily dissociates from the apoenzyme it is regarded as a coenzyme. Coenzymes are mainly derivatives of water-soluble vitamins. In the conjugated enzymes neither cofactor nor protein compounds taken saparately exhibit the catalytic activity.

Enzymes Functional OrganizationThe protein nature of enzymes determines their functional properties. First of all the enzymes are macromolecules substantially exceeding the reagents, or substrates, in size. That is why not the entire surface of the enzyme does actually work but only a part of it – the active centre.

Active centre is an area of enzyme molecule (unique combination of amino acid residues) that directly takes part in the catalysis. These amino acids are located in different positions in polypeptide chain but due to the steric packing (formation of tertiary structure) they come closer creating the active centre. Thus the active centre is formed at the level of tertiary structure. That is why denaturation leads to the loss of catalytic activity.

simple enzymes

apoenzyme cofactor

enzymes

The substance transformed by enzyme (E) is named substrate (S). The substance formed by enzyme is a product (P).

S РЕ

complex enzymes

5

The functional groups of the enzymes active centres:• СООН – of dicarboxylic amino acids;• amino groups of lysine and terminal phenol groups of tyrosine;• ОН-groups of serine and threonine;• SH-groups of cysteine and disulfide bridges of cystine;• indole groups of tryptophan;• guanidine - of arginine;• imidazole – of histidine;• thioether – of methionine;• hydrophobic – of aliphatic amino acids• aromatic ring – of phenylalanine.In conjugated enzymes the non-protein part is involved into the active centre. The following areas are found in the active centre:•substrate binding site: it is responsible for recognizing and binding with the substrate resulting in the formation of the enzyme-substrate complex (ES); •catalytic site: it directly performs the catalysis.The first model of active site proposed by E. Fisher stated that enzyme and substrate fit each other similar to lock and key. It means that the enzyme-substrate complex (ES) is a rigid structure. But in the protein molecule the rigid areas alternate with flexible ones, that is why preferential is the theory of induced fit proposed later by D. Koshland. This theory implies the flexibility of the active site. The substrate induces the conformational changes of enzymes active centre in such a way that precise orientation of catalytic groups is provided. The enzyme may force substrate to fit in, too.

Enzymes Properties1. Effect of Temperature on Enzymes Activity (Thermolability)

The rate of enzyme reaction increases when the temperature of the medium is increased in the certain interval as the kinetic energy of the molecules rises. At the optimal temperature (40◦С) the enzymatic reaction rate is maximal. With the further temperature rise the enzyme denaturation occurs and the reaction stops.

S

E ES

t°

v

40

S u b s r a t e

6

3. Enzymes Action Specificity

А. Specificity of the Transformation Way. The functional groups of the active centre are able to perform a chemical transformation of the substrate only into the certain product. For example, the same substrate (amino acid histidine) may be converted into different products such as ketonic acid or amine depending on the kind of enzyme: histidase or decarboxylase, respectively.

В.Substrate Specificity. It means the enzyme ability to bind a certain substrate due to the unique structure of the active centre. There are two types of substrate specificity: 1.Absolute specificity: it is the ability of the enzyme to catalyze the transformation of a peculiar substrate. For example, arginase breaks down only arginine. 2.Relative specificity: it is the ability of the enzyme to catalyze only one-type changes of substrates of similar structure. For example, lipase catalyzes the lipolysis (lipid hydrolysis), pepsin – proteolysis (protein hydrolysis). 3.Stereospecificity: it is the ability of the enzyme to act only on a certain stereoisomer. For example, oxidase of D-amino acids cannot convert their isomers L-amino acids.

Histidine

Ketonic acidHistamine

NН3СО2

decarboxylasehistidase

This effect is determined by several factors: enzyme denaturation under extremely high and low pH values;changing of charge value of substrate or enzyme.

As there are groups in the active centre that can be ionized thechange of pH leads to the change of functional groups ionization degree that may cause conformational changes of the enzyme molecule and therefore it leads to the another catalytic activity.

2. Effect of pH on Enzymatic Reaction Rate

v

рН

7

ISOENZYMES

Multienzyme Complexes

They are supramolecular complexes consisting of various enzymes sequentially catalyzing different stages of the same process. These systems work as a kind of conveyor: the product of the first reaction is the substrate for the second reaction, and so on. It results in space and time advantages. In many cases these enzymatic ensembles are bound to the certain cellular organelle or membrane. Example: pyruvate dehydrogenase complex, fatty acid synthase .

Multiple molecular forms of enzymes, or isoenzymes, are enzymatic proteins differing from each other in physicochemical properties but catalyzing the same reaction. These differences of physicochemical properties in isoenzymes are genetically determined. The amount of certain isoenzymes varies in different cells, tissues and organs. This feature makes them useful for differential diagnostics of these organs lesions.

Example: lactate dehydrogenase(LDH) is a complex protein with quaternary structure presented by four subunits of two types: Н(heart) and М (muscle). The combination of these subunits (polypeptide chains) gives five isoenzymes.

These isoenzymes have different motility under electric field that is why they can be separated by electrophoresis. They are organo-specific, id est located in certain organs: LDH1 – mainly in heart, LDH5 –predomainly in skeletal muscles. In norm their concentration in blood is minimal. As the result of the organ destruction (myocardium infarction) the LDH1

concentration in blood rises drastically. These isoenzymes are named indicatory enzymes.

Days after infarction

LDH2

Н

Н

НМ

LDH3

Н НМ М

LDH5

М ММ М

LDH1

Н

Н

Н

Н

LDH4

ММ МН

LDH1

Е1

S

P

Е2

Е3

Е4

matrix

membrane

8

General Information About Catalysis

•Difference between the free energy of the substrates and the reaction products determines the possibility of the reaction. •If the substrates have more free energy than the products a spontaneous reaction is possible (exergonic reaction). Contrariwise, the endergonic reactions consume energy to proceed. •The exergonic reactions rate depends on the energy barrier, or activation energywhich is the additional energy portion (for example, heat) given to the molecules of the substrates to make them possible to react. •The catalyst (enzyme) does not affect the free energy of both initial substances and products but it does affect the energy profile. It means that in the presence of the catalyst the reaction proceeds via low energy barrier. •Thus enzymes decrease the reaction activation energy that results in the extreme increase of the enzymatic reaction rate compared with the non-enzymatic one.

8

non-enzymatic reaction

enzymatic reaction

activation energyof non-enzymatic reaction

activation energyof enzymatic reaction

reaction path

free

ene

rgy

final state

initial state

S

P

transition state

9

Mechanism of the Enzymes Action

In the classic works of Michaelis and Menten the enzymatic catalytic process is divided into three stages:

E+S ES ES* EP E+P

•1. Binding of the substrate to the enzyme and formation of the enzyme–substrate (ES )complex. This stage proceeds rapidly and reversibly. Thereby the exact dimensional, geometrical and electrostatic complementarity between the enzyme active centre and the substrate is achieved. •2. The transformation of the primary ES-complex to the activated one (ЕS*) and formation of the enzyme-product (EP) complex. This stage occurs slowly and it is accompanied by theloosening of the substrate bonds by means of the enzyme functional groups. The activation energy of the reaction decreases. •3. Dissociation of the reaction products from the active centre (E + P) and their diffusion into the surrounding medium. The enzyme is not a component of the final products.

The effectiveness of enzymatic catalysis is associated with the following molecular effects:

reactants orientation The substrate binds the enzyme at least in at three points (three-point attachment) that causes the high affinity (complementarity) of binding and the substrate orientation. It results in hundred- and thousandfold increase of the reaction rate which is impossible in the spontaneous interactions.

tension and deformation While binding to the active centre of the enzyme the substrate molecule undergoes deformation. Tension and strain points break down easily.

acid-base catalysisThe active centres of the enzymes contain both acidic and alkaline groups so they may work both as donors and acceptors of protons. This property is not characteristic of the inorganic catalysts. It facilitates the reorganization and disruption of the substrate bonds. For example the enzymes containing histidine residues in their active centres work this way.

covalent catalysisThe active centres of certain enzymes contain groups able to create the covalent bonds with the substrates leading to the following formation of the unstable ES-complexes that release the reaction products easily.

Generally enzymes use the combination of the above mentioned mechanisms that provide their phenomenal activity.

k1 k2

k-1

10

Enzymatic Reactions Kinetics

It studies the effect of different factors on the velocity of the enzyme catalyzed reaction.

• “To study kinetics without understanding the reaction mechanism is as good as to find the hat and to lose its owner” (Ingold).

•The enzymatic reactions rate depends on such factors as temperature, pH of the reaction milieu, the enzyme concentration and substrate concentration.

КM – Michaelis constant – corresponds to the substrate concentration at half-maximal rate (КM is expressed in moles/L).

Effect of Substrate Concentration

If the rate (v) is plotted against the substrate concentration [S] a hyperbola is obtained. In case of the low substrate concentration this dependence is linear but the curve flattens afterwards. The high substrate concentration corresponds to the maximal velocity vmax. Further increase of the substrate concentration does not affect the reaction rate, because the saturation of the enzyme by its substrate is observed.

V =Vmax . [S]КM + [S]

КM is determined experimentally. It is helpful for the understanding of various effectors'(activators and inhibitors) action on the enzyme activity. Km value is inversely related to the enzymes activity.

Michaelis equation describes the dependen-ce of the enzymatic reaction rate on the substrate concentration.

More useful for results processing is the Lineweaver-Burk plot. This graph reveales a directly proportional dependence helpful in obtaining the Michaelis constant value easily and therefore evaluating the enzymes activity in certain conditions.

Substrate concentration [S]

Rea

ctio

n r

ate

V

11

Enzyme Activity Regulation

Enzyme activity is assessed by the substrate consumption or product accumulation in a certain period of time. Enzyme activity is measured under the standard conditions:• temperature 25°С;• рH optimal value;• saturating substrate concentration (reaction rate should be maximal);• the conjugated enzyme should be saturated with its cofactor.International enzymatic activity unit means the enzyme amount able to convert1 micromole of a substrate in 1 minute under the standard conditions. Specific activity equals the enzyme amount in the sample divided by the protein weight in it. It is expressed in micromoles/(min x mg). Katal (kat) means the enzyme amount able to convert 1 mole of a substrate in 1 second under the standard conditions.

Factors influencing enzyme activity: • temperature• рH• enzyme concentration• substrate concentration• presence of activators or inhibitors

Thus enzymes are catalysts with regulated activity that is important for homeostasis maintenance.

Enzymes Activation

1. Allosteric RegulationMany enzymes are allosteric, or regulatory ones. Beside the active centre they have an allosteric centre located in the alternative site of an enzyme molecule. Such enzyme usually consists of several subunits, or it has a quaternary structure. The allosteric centres serve for binding low-molecular substances – allosteric effectors. The effectors' structure has to be complementary to the allosteric centre like the substrate structure is complementary to the enzyme active centre. Allosteric effectors may be either activators or inhibitors. The allosteric regulators bound to the regulatory subunits result in the change of the enzyme conformation. The latter induces the conformational change of the catalytic subunit resulting in increase (allosteric activation) or decrease (allosteric inhibition) of the enzyme activity.

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•Heterotropic allosteric effectors are substances different from the substrate in their chemical structure.

•Homotropic allosteric regulation is the characteristic of enzymes consisting of identical protomers each having an active centre. In this case the substrate works as an effector: after binding with one protomer it activates others.

2. Activation by CofactorsThe presence of cofactor is obligatory for the conjugative enzymes to be active. The active forms of vitamins and bi- (or mono-)valent metals as a rule serve as cofactors. About 25 percent of enzymes need metals for activation. The activation is performed by several mechanisms:

metal ions take part in the formation and stabilization of the active centre; metal ions work as prosthetic groups;metal ions facilitate substrates binding to an active centre; metal ions bind the substrate forming the metal-substrate complex that is

considered to be true substrate which may be converted by the enzyme.

Some metals–

cofactorsCu2+

Fe2+

Fe3+

K+

Mg2+

Mn2+

Mo+

Ni2+

Se+

Zn2+

active centre

allosteric centre

allosteric activation

activator inhibitorsubstrate

allosteric inhibition

13

4. Enzymes Phosphorylation/DephosphorylationSome enzymes are activated by means of phosphorylation (addition of the negatively charged phosphate group) by enzyme proteinkinase.

3. Partial Proteolysis means the transformation of an inactive enzyme (zymogen or proenzyme) to an active one by means of polypeptide chain cleavage. In this case one peptide bond is broken down, id est the enzyme primary structure alteration takes place. This type of activation is a characteristic of protelytic enzymes taking part in proteins digestion.

This mechanism was formed evolutionarily and it is essential for proteins protection from proteases action.

proteinkinase

АTP АDP

lipase lipase Pinactive active

5. Protomers Association and Dissociation Some enzymes may be activated by addition or removal of the regulatory subunits or protomers. In this case the quaternary structure alteration of the enzyme molecule takes place.

glycogen synthase

glycogensynthase

inactiveactive

proteinphosphataseН2О

association

dissociation

Other enzymes are activated due to dephosphorylation:

неактивный ферментполипептидный участок

активный ферментнеактивныполипептидный участок

активный ферментpolypeptide chainactive enzyme

Н2О

inactive enzyme

14

Learning of the enzymes inhibition makes it possible to understand the mechanism of the enzymes action, the essence of catalysis as well as to study various metabolic processes. Also the enzyme inhibitors are used as remedies. Inhibitors are characterized by the affinity to the enzymes.In acccordance with this characteristic they are divided into two groups: 1.Irreversible inhibitors: they are the compounds specifically bindidng the functional groups of the enzymes by means of stable covalent bonds formation.2. Reversible inhibitors: they form the unstable non-covalent bonds with the enzymes and may easily dissociate from them.

Action of various drugs is based on the competitive inhibition.It is well-known that gout is associated with the accumulation of uric acid in joints. This disease is treated by allopurinol that is the competitive inhibitor of xanthine oxidase. This is the key enzyme of uric acid formation process.

irreversible

inhibitors

competitive inhibitors

non-competitiveinhibitors

reversible

Substrate (SS) and inhibitor (I) are similar in their chemical structure and compete for the enzyme (E) active centre.It is possible to eliminate such inhibitor by addition of high amounts of the substrateb into the medium.

1. Competitive Inhibition

Е

Е

Е

purine bases hypoxantine uric acidxantine oxidase

allopurinol

_

Enzymes Inhibition

15

Occasionally under the continuous action of non-competitive inhibitor and formation of the stable bonds with the enzyme such inhibition may become irreversible.Irreversible inhibitors are usually toxic. For example, cyanides form tight bonds with iron atom from the active centre of cytochrome oxidase and in this way cyanides block the mitochondrial respiratory chain leading to the cell death. • Heavy metals ions are very toxic as they block SH-groups in the active centres of the enzymes. Thereby the substrate may be binded to the active centre of the enzyme but it does not lead to products formation.• Acetylcholine esterase inhibitors irreversibly phosphorylate the enzyme catalytic group (poisonous substances sarin, soman).

2. Non-competitive Inhibition

Non-competitive inhibitor is structurally different from the substrate (S), or it does not have any affinity to free enzyme. That is why it usually binds the enzyme not in the active centre but in another site of the enzyme molecule. In this case the formation of a triple complex ESI may occur. It is impossible to eliminate the non-competitive inhibition by increasing the concentration of substrate, for this purpose the substances binding the inhibitor are needed.

Е Е

Е

Non-competitive inhibitor decreasesVmax but does not change KM

Allosteric inhibition is related to the non-competitive type. It is usually realized by negative feedback regulation, that means that the final product (Рn) of the certain reactions sequence is the inhibitor of the enzyme catalyzing the first reaction (E1). In this case the regulatory allosteric enzyme catalyzing the rate-limiting reaction that is the slowest will be inhibited:

S Р1 Р2 Р3Е1 Е2 Е3 Еn

_

Рn

Competitive inhibitor increases KM but does not change Vmax

16

Medical Importance of Enzymes

Enzyme pathology is the study of enzymes activity in normal and pathological states including the diagnostics of hereditary diseases associated with genes defects. Such defects cause certain proteins synthesis failure. If these proteins are enzymes it leads to the catalyzed reactions rate alteration and the metabolism disturbance.

Enzyme therapy means the usage of enzymes and regulators of enzymes activity as drugs. Proteolytic enzymes inhibitors normally produced by the pancreatic gland in inactive state are used for pancreas autolysis prevention in case of acute pancreatitis.

Enzyme diagnostics is developed in the following directions:

1) use of highly purified enzymes as analytic agents or selective reagents for the quantitative analysis of normal and pathological chemical substances in biological fluids for the diagnostic purpose. This method is characterized by high specificity and sensitivity. It is used for measuring of glucose, lactate, cholesterol, triacylglycerides, urea, uric acid level and other substances.

2) detection of the enzymes activity in blood in case of organs and tissues damage. The change of enzymes level is very likely the result but not the cause of pathological process. Such investigations are usually conducted in blood plasma. However the enzymatic analysis of other biological fluids such as urine, pancreatic juice may also give the useful information. A lot of enzymes detected in blood plasma are intracellular so they are released into blood because of the cell membranes damage. These enzymes are called “indicatory enzymes”. Intracellular enzymes are present in blood in low amounts as the result of cells renewal. At the same time there are various enzymes, for example, rennin, blood coagulation factors and others that are actively secreted into blood for their physiological functions.

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Test 1Choose the enzyme with the absolute specificity.A. EsteraseB. AmylaseC. PepsinD. UreaseE. Trypsin

Test 2 Enzymes are classified by:A. Chemical structureB. Metal atoms included in their cofactorsC. Coenzymes typeD. Apoenzymes structureE. Type of the reaction they catalyze

Test 3 Which class of enzymes does pyruvate kinase belong to?A. LigasesB. HydrolasesC. OxidoreductasesD. TransferasesE. Isomerases

Test 4The area of an enzyme molecule formed by the amino acids residues responsible for the binding with its substrate and directly taking part in catalysis is named ...A. Allosteric centreB. Active centreC. CoenzymeD. CofactorE. Prosthetic group

Test 5 Human enzymes show the maximal activity under:A. Room temperature (25°C)B. 70°CC. 37°CD. 84°CE. >100°C

KEYS: 1 – D; 2 – E; 3-D; 4 – B; 5 – C.

Multiple Choice Questions

18

VITAMINS”Life amines” (K. Funk)

Vitamins –essential for normal vital functions alimentary low-molecular organic substances restrictly synthesized or not synthesized in human organism. Vitamin sources for the humans are food and intestinal flora. Some vitamins come to organism in the form of precursors – provitamins transforming in tissues to active substances (e.g. carotenoids contained in carrots, tomatoes and paprika are provitaminsof vitamin A).

Vitamins have specific features:they are not structural components of organs or tissues;they are not used as energy sources.

Classification and nomenclature

For designation of each vitamin there are literal marking, chemical and physiological names:

vitamin C = ascorbic acid = antiscorbutic vitaminVitamins are classified according to their solubility in water and in fats.

1. Fat-soluble vitamins - vitamins A, D, E, K and vitamin-like substances – ubiquinone(vitamin Q), essential fatty acids (vitamin F).

2. Water-soluble vitamins – vitamins of B group, H, C, vitamin-like substances – choline, lipoic acid, carnitine, orotic acid and others.

Vitamin Imbalance in the Organism

Hypovitaminosis means the certain vitamin deficiency that is clinically and biochemically manifested.

Avitaminosis is the full absence of vitamin in diet or the full disturbance of this vitamin usage in the body.

Hypervitaminosis is the vitamin excess. This state is related to fat-soluble vitamins which may be accumulated in the body resulting in a toxic effect.

Causes of vitamins imbalance:exogenous: improper diet. endogenous: absorption defect;excessive need (for example, in pregnancy);liver diseases;increased breakdown of vitamins ;intestinal microflora deficiency.

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Hypovitaminosis is usually non-specific state. It is manifested as the increased fatigability and the decreased immunity whilst avitaminosis has specific clinical manifestations associated with disturbance of certain biochemical processes. The reason is that almost all water-soluble vitamins are transformed in organism to the active coenzyme form (usually nucleotide or phosphoric ester) with the following binding with the protein compound (apoenzyme) thus forming a complex holoenzyme which catalyzes certain reaction:

bindingvitamin coenzyme apoenzyme

holoenzyme

productsubstrate

activation

catalysis

Vitamins MetabolismAfter the intestinal absorption fat-soluble vitamins are accumulated in tissues where they are utilized as the need arises. Water-soluble vitamins are transformed to their active forms, or coenzymes. After performing their catalytic function vitamins are broken down and excreted.

Antivitamins

Antivitamins are natural and synthetic compounds inhibiting the action of vitamins. They may be divided into two groups:- vitamins structural analogues which may block the active centres of enzymes by means of binding with the protein compound instead of vitamin. It results in the competitive inhibition of this enzyme and in certain biochemical reaction disturbance with the following hypo- or avitaminosis manifestation. For example, isoniazid that is the drug for tuberculosis treatment, is the antivitamin for vitamins PP and В6.- non-specific antivitamins which break down vitamins or bind with them. For example, raw fish contains enzyme thiaminase which cleaves thiamine (vitamin B1). The uncooked egg white contains avidin – antivitamin H.

Biological Role of Vitamins

Water-soluble vitamins usually play the role of coenzymes of complex enzymes.

Fat-soluble vitamins are the components of biological membranes. They perform certain regulatory functions. For example, they work as antioxidants and protect biological molecules from the action of reactive oxygen types.

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VITAMIN B1 – THIAMINE – ANTINEURITIC VITAMIN

B1

АТP AMP

ТPP

thiamine phosphokinase

Biological Role of TPP

• a component of pyruvate dehydrogenase and α-ketoglutarate dehydrogenase complexes catalyzing the reactions of oxidative decarboxylation of pyruvate and α-ketoglutarate (Krebs cycle), i.е. promotes energy formation from carbohydrates and lipids:

pyruvate acetyl-CoA + СО2

ά-ketoglutarate succinyl-CoA + СО2

• a component of transketolase (pentose phosphate pathway of glucose oxidation) essential for fats and nucleic acids synthesis.

Hypovitaminosis: polyneuritis (beri-beri, Wernicke-Korsakoff syndrom) –cardiovascular, nervous system and gastrointestinal tract affection. Biochemically it is characterized by the increase of pyruvate concentration in blood and tissues.

Coenzyme form – thiamine pyrophosphate - TPP (cocarboxylase)

Vitamin is activated by ATP (as a source of two phosphate residues) and enzyme thiamine phosphokinase:

pyruvate dehydrogenase

ά-ketoglutarate dehydrogenase

+

21

VITAMIN В2 – RIBOFLAVIN – GROWTH VITAMIN

Coenzyme forms:

flavine mononucleotide - FMN

flavine adenine dinucleotide – FAD

B2

Vitamin is activated by ATP as a donor of phosphate (FMN synthesis) and AMP (FAD synthesis):

Biological Role of FMN and FAD

FMN and FAD are prosthetic groups of a large number of enzymes – flavoproteins (both aerobic and anaerobic dehydrogenases and oxidases) taking part in oxidation of different intermediates of carbohydrate, lipid and protein metabolism.

Hypovitaminosis: growth retardation, inflammation of tongue mucouse membrane (glossitis), lips, skin epithelium, eyes (conjunctivitis, keratitis, cataract).

FMN

FAD

ATP PP

Н

ATP PP

Riboflavin is a derivative of tricyclicisoalloxazine and pentabasic alcohol ribitol

isoalloxazine ring

ribitol

22

Biological Role of NAD+, NADP+

as coenzymes of pyridine-dependent anaerobic dehydrogenases they perform hydrogen transfer in the oxidation-reduction reactions;

they are the substrates for synthetic reactions of replication and reparation;

they are allosteric regulators of certain enzymes activity.

VITAMIN В3 – РР (PELLAGRA PREVENTING) – NIACIN (nicotinic acid + nicotinamide)

Coenzyme forms:

nicotinamide adenine dinucleotide – NAD+

nicotinamide adenine dinucleotide phosphate - NADP+

Hypovitaminosis – pellagra:

dermatitis (symmetric skin lesions);

diarrhea (pathological changes of oral and intestinal mucosa);

dementia (mental retardation).

NicotinamideNicotinic acid

As distinct from other vitamins nicotinamide may be synthesized in organism from amino acid tryptophan.

NAD+

+

NADP+

+

23

pyridoxine

Coenzyme forms:

pyridoxal phosphate - PALP

pyridoxamine phosphate –PAMP

Biological Role of Pyridoxal Coenzymes

PALP and PAMP are the components of pyridoxal enzymes catalyzing the main reactions of nitrogen (protein) metabolism:

transamination reactions (transaminases or aminotransferases);

reactions of amino acids decarboxylation (decarboxylases);

heme and sphingolipids biosynthesis;

transformation of tryptophan to vitamin PP;

active transport of amino acids through the cell membrane.

Hypovitaminosis: pellagra-like dermatitis (it is not sensitive to treatment with vitamin PP), anemia, spasms;

pyridoxal dependent enzymopathies (homocysteinuria),

tuberculosis treatment with isoniazid (antivitamin В6)

Coenzyme forms are synthesized by means of vitamin phosphorylation using ATP and enzyme pyridoxal kinase:

ATP ADP

PALPB6 Pyridoxal kinase

VITAMIN В6

pyridoxal pirodoxamine

PALP PAMP

-О-РО3Н2-О-РО3Н2

24

Coenzyme form – tetrahydrofolic acid (THFA) – Н4-folate

Biological role of THFA: it takes part in the intermolecular transfer (from donor molecule to acceptor) of one-carbon fragments (which join to the 5-th or 10-th atom of THFA) in the processes of purine and pyrimidine nucleotides biosynthesis (i.e. DNA and RNA synthesis), amino acids formation (methionine, serine, glycine), synthesis of various physiologically active compounds.

Hypovitaminosis causes macrocytic megaloblastic anemia characterized by inhibition of nucleic acids synthesis and erythrocytes formation, appearence of large erythrocytes (macrocytes) and immature erythrocytes (megaloblasts) in blood. Also sprue (chronic enteritis) and decreased amount of plateletes and leukocytes are typical.

Hypovitaminosis may be caused by disbacteriosis associated with long-term antibacterial therapy with sulfonamides – structural analogues of p-aminobenzoic acid that is the compound of folic acid). In bacterial cells these drugs inhibit folic acid formation essential for bacterial DNA and RNA synthesis.

FOLIC ACID (VITAMIN В9 or Вс) – GROWTH FACTOR (ANTIANEMIC)

Two NADPH molecules are used for tetrahydrofolic acid (THFA) formation. The first two hydrogen atoms are attached to the 5-th and to the 6-th carbon atoms in pteridine ring resulting in formation of dihydrofolicacid (DHFA). Another two hydrogen atoms are binded to the 7-th and to the 8-th carbon atoms in pteridinering resulting in formation of tetrahydrofolic acid (THFA).

DHFA + NADPН2 THFA + NADP+dihydrofolate reductase

FA + NADPH2 DHFA + NADP+folate reductase

One-carbon fragments:methyl -СН3; methylene -СН2-; methenyl =СН-; formyl –СНО; formimine –СН=NНetc.

Vitamin sources: food and intestinal microflora

Biochemical functions of folic acid are associated with vitamin В12 metabolism. These vitamins are synergists.

p-aminobenzoicacid

glutamic acidpteridine ring

4-aminopterin, methotrexat – competitive inhibitors of dihydrofolate reductase used as anticancer drugs

25

VITAMIN В12 - COBALAMINE – ANTIANEMIC

Biological Role

methyl-В12 takes part in transmethylation reactions together with THFA (the intermolecular methyl groups transfer). These reactions are important for methionineresgeneration from homocysteine, biosynthesis of epinephrine, creatine, purine and pyrimidine bases that are used in DNA and RNA synthesis.

DА - В12 takes part in hydrogen atoms transfer in isomerization reactions. It acts as a coenzyme of methylmalonyl-CoA mutase catalyzing the transformation of methylmalonyl-CoA (formed from the branched-chain amino acids) to succinyl-CoA (Krebs cycle metabolite).

Hypovitaminosis is megaloblastic, or pernicious, anemia (Addison-Birmer disease). It is developed due to the inhibition of nucleic acids synthesis in tissues with active cell division, for example, in bone marrow.

Endogenous hypovitaminosis results from disturbance of cobalamine absorption in the digestive tract. It may occur because of glycoprotein transcorine (intrinsic factor) lack that is necessary for B12 (extrinsic factor) absorption.

Coenzyme forms: methylcobalamine (methyl-В12)desoxyadenosylcobalamine (DA - В12)

Vitamin sources: animal food and intestinal microflora. Unlike other vitamins B12 is synthesized only by microorganisms.

В12 is the only vitamin containing metal ion – cobalt.

26

VITAMIN С – ASCORBIC ACIDANTISCORBUTIC VITAMIN

Avitaminosis – scurvy (scorbutus). It is characterized by collagen and chondroitin sulfate synthesis impairment leading to connective tissue atrophy, anemia, swollen gums and loose teeth.

L-ascorbic acid L-dehydroascorbic acid

Biological Role

takes part in oxidation – reduction reactions;

takes part in hydroxylation reactions as a strong reducing agent: in the synthesis of neurotransmitters, steroid hormones and hemoglobin, in collagen posttranslational modifications (hydroxylation of proline and lysine residues);

enhances iron absorption and utilization;

is an antioxidant.

VITAMIN Р - RUTIN - PERMEABILITY VITAMIN

Vitamin P contains the substances that are plant pigments - flavonoids.

Biological Role of Favonoids

they reduce permeability of blood vessels, especially capillaries. Vitamin P prevents hyaluronic acid (the basic compound of the extracellular matrix) from degradation by inhibition of enzyme hyaluronidase;

They prompote maintaining of ascorbic acid in the reduced state.

Hypovitaminosis is characterized by the increased permeability of blood vessels(bleeding, hemorrhages), fatigability, limb pains. It is combined with vitamin C deficiency. Ascorutin, that is the drug containing vitamins C and P, is used to decrease the permeability of blood vessels.

Oxidized form is unstable, it easily breaks down loosing its biological activity.

27

Hypovitaminosis occurs in the case of normal intestinal microflora deficiency (long-term antibacterial therapy). Experimentally it was developed in animals continuously fed with uncooked eggs containing avidin – the protein inhibiting biotin absorption. The symptoms of hypovitaminosis are seborrhea-like skin lesions and hair loss.

VITAMIN В5 – PANTOTHENIC ACID

Biological RoleНS-CoA takes part in:

acetate and fatty acids activation;fatty acids oxidation and synthesis;cholesterol and other steroids synthesis; ketone bodies synthesis;acylation process in heme and acetylcholine synthesis;

oxidative decarboxylation of pyruvate and α-ketoglutarate.

VITAMIN H – BIOTIN – ANTISEBORRHEIC VITAMIN

Sources: intestinal bacteria, plant and animal food

Biological role: as a prosthetic group it is incorporated into biotin-containing enzymes catalyzing carboxylation and transcarboxylation reactions such as:

fatty acids synthesis (acethyl-CoA-carboxylase);

transformation of pyruvate to oxaloacetate (pyruvate carboxylase);

purine nucleotides synthesis.

Hypovitaminosisis not found

(Greek: pantos –widespread)

β-mercaptoethylamine pantothenic acid phosphoadenosine diphosphate

Coenzyme forms:НS-CoA – coenzyme А or acylation coenzyme4-phosphopanthotein

НS

β-alanin

HS-CoA

28

Test 1Hydroxyproline is an essential amino acid in collagen structure. Which of the following vitamins

takes part in the formation of this amino acid by proline hydroxylation?A. Riboflavin

B. Thiamine

C. Ascorbic acid

D. Pyridoxine

E. Folic acid

Test 2Which vitamin coenzyme is used for transfer of one-carbon atom fragments?A. Folic acid

B. Biotin

C. CobalamineD. PyridoxineE. Vitamin P

Test 3Pernicious anemia is a pathological state caused by the deficiency of vitamin B12. Which chemical element is a constituent of the structure of this vitamin?A. Iron

B. Molybdenum

C. Zinc

D. Cobalt

E. Magnesium

Test 4Which substance is accumulated in blood in beri-beri?A. Urea

B. Pyruvate

C. Succinate

D. Citrate

E. Malate

5. A patient complains about dermatitis, diarrhea and dementia. Which vitamin deficiency is the cause of such pathological state?A. Folic acid

B. Ascorbic acid

C. Niacin

D. Biotin

E. Rutin

KEYS: 1 – C; 2 – A; 3 – D; 4 – B; 5 – C.

Multiple Choice Questions

29

Catabolic pathways release free energy asATP, NADH, NADPH, and FADH2.This energy can be used for anabolic

conversion of simple molecules into cellular macromolecules.

Energy-containingnutrients:

carbohydrateslipids

proteins

Energy-poorEnd products

СО2Н2ОNН3

CatabolismAnabolism

Cellular macromolecules:proteins

polysaccharideslipids

nucleic acids

Molecules-precursors:amino acids

monosaccharidesfatty acids

nitrogenous bases

Chemical energy

АDP+P

АТPNADН+

NADPН+

FADН2

NAD+

NADP+

FAD

Coupling of Exergonic and Endergonic reactions

There are two groups of biochemical reactions in living cells: one group, or exergonic reactions, results in energy liberation from the bioorganic molecules, another one consumes energy for different biochemical and physiological processes such as synthesis of different substances (endergonic reactions), nerve impulse conduction, active transport and muscle contraction. Exergonic and endergonic reactions are coupled.

Exergonic reactions are usually catabolic ones. They are characterized by cleavage and oxidation of "fuel" molecules (energy-containing nutrients) that are food molecules.

Endergonic reactions are anabolic ones. They are reations of different bioorganic compounds, or cellular macromolecules, synthesis.The ensemble of all biochemical conversions of chemical substances in living cells is called metabolism.

ENERGY METABOLISM

30

The main high-energy compound in living organisms is adenosine triphosphate (ATP), which provides transfer of free energy fromendergonic processes to exergonic ones. ( ) indicates that the transfer of the group, which is attached by such bond, isaccompanied by yield of a large amount of free energy.The free energy of hydrolysis of the terminal phosphate group of ATP is a

= - 30.5 kJ / mol

Synthesis of ATP from ADP and phosphate , or ADP phosphorylation, occurs in the human body in two ways differ in the source of energy which is needed for the formation of high-energy bond:

In order to pair these processes a mediator is usually needed. In exergonic reactionscompounds with high energy potential ~ E are synthesized. They are subsequently used in endergonic reactions. This way chemical energy is transferred from exergonic to endergonicprocesses. Substances with a high energy potential, or high-energy substances (~ E), are biological molecules that have a high standard free energy level of terminal phosphate grouptransfer.

ribose adenine

ribose аdenine

hydrolysis

АDP

АТP

P

~

∆G0

АDP + P АТP

energy

1. Oxidative phosphorylation is the main pathway of ATP synthesis. It occurs due to the energy of oxidation of various substances such as metabolites, or substrates for oxidation.

2. Substrate phosphorylation takes place due to the energy of the cleavage of high-energy substrate bonds.

In addition to ATP, there are another high-energy compounds such asmetabolites of carbohydrate, lipid and amino acid metabolism, as well as phosphogen (creatine phosphate), which act as a reservoirs of high-energy bonds:

High-energy compounds:

∼ phosphoenolpyruvate - 61.9 kJ / mol∼ carbamoyl phosphate - 51, 4 kJ / mol∼ 1,3-bisphosphoglycerate - 49.3 kJ / mol

∼ creatine phosphate - 43.1 kJ / mol

∆G0

~

31

It is important to understand that you can not extract energy directly from the nutrients coming from food, but from their degradation products which are formed during metabolism.

Metabolism in the human body passes of the following stages:1. Supply of nutrients (proteins, fats, carbohydrates, vitamins,

minerals and water which come with food).2. Digestion of nutrients in the digestive tract into simple substances that can be

absorbed by the intestinal mucosa.3. Biotransport of digestive products in blood and lymph. Their penetration

through the membranes of blood vessels and cells to specific organs and tissues.4. Intracellular metabolism, or the intermediate metabolism.

5. Secretion of the end products of metabolism:carbon dioxide, water, urea, etc., from the body

Thus, the accumulation of energy in the specific phosphate bonds of ATP is the basis of energy transfer mechanism in the living cells. But the living cell is a non-equilibrium system, so it is possible to accumulate chemical energy in the high-energy bonds.

Stages of Biomolecules CatabolismStage 1 (specific) is the preparatory one . Macromolecules of carbohydrates, proteins, lipids are splitted into simple monomers. This stage is hydrolytic and represents digestion in the gastrointestinal tract. Such reactions are not accompanied by a significant release of energy (up to 1% of energy substrates is released in the form of heat).Stage 2 (specific). Enzymatic cleavage reactions of substances formed in the first stage take place. A certain amount of energy (up to 20%), which is partially released in the form of heat, or accumulated in ATP bonds, is formed. Reactions in this stage occur in anaerobic conditions inside the cells (in cytoplasm and partially in mitochondria).The main processes of this stage are:for monosaccharides - glycolysis, resulting in the formation of pyruvate, and then

acetyl-CoA;.for fatty acids - the beta-oxidation, the end product of which is acetyl CoA;for glycerol - conversion to pyruvate and then to acetyl CoA;for amino acids and nucleotides - deamination with the release of ammonia and

final conversion of nitrogen-free carbon skeletons intoacetyl CoA.

Thus, the acetyl CoA is the end productof the second stage of catabolism.

32

acetyl-CоА

pyruvate

Stage 3 is common to all types of metabolism. At this stage oxidation of acetyl-CoA to the end metabolites such as carbon dioxide and water takes place. This stage occurs in the mitochondria which are power stations for cells. It consists of two processes:1. Citric acid cycle (CA cycle), resulting in the formation of CO2, hydrogen atoms being

used to restore NAD and FAD coenzymes. Hydrogen is a universal energy fuel, which is used in the respiratory chain to form energy and water.2. System of electron transport in the membranes of mitochondria, where hydrogen atoms

are transferred onto oxygen to form water. This system is associated with oxidative phosphorylation, in which the energy of biological oxidation is used to synthesize ATP.

Lipids

monosaccharides glycerol fatty acids

CAC

heat АDP + P АТP

О2 Н2О

2Н+ 2Н+ 2Н+ 2Н+

Mitochondrial respiratory chainО2

Proteins Carbohydrates

aminoacids

СО2

СО2СО2

energy

33

•Biological oxidation which is accompanied by the consumption of oxygen and the formation of energy, water and carbon dioxide is called tissue respiration. This is a multi-step process of hydrogen transfer (protons and electrons) from the oxidation substrates through a series of intermediate carriers to oxygen in order to form water and release energy.

•Substrates of oxidation are formed during the catabolism of proteins, fat and carbohydrates. These are substances that undergo dehydrogenation, i.e. elimination of hydrogen atoms.

•Hydrogen is a universal energy fuel, which is used in tissue respiration to form energy and water.

•Tissue respiration is represented as a multienzymechain of electrons transfer (ETC), which is called the mitochondrial respiratory chain or respiratory ensemble. It structurally organized, as its components are built into the inner membrane of mitochondria (5 to 20 thousand ensembles in a single mitochondrion).

BIOLOGICAL OXIDATION

Intermediate carriers (P) are used in the transport of electrons from the electron donor source (substrate) to the terminal acceptor oxygen.

The complete process is a chain of successive oxidation-reduction reactions, in which interaction between carriers takes place. Each intermediate carrier (P) initially acts as an acceptor of electrons and protons. It leads to the reduction of the carrier. Then it transfers an electron to the next vector and returns to the oxidized state. At the last stage the carrier transfers electrons to oxygen, which later is reduced to water by adding protons.

SH2 О2

SH2

Н2

outer membrane

inner membrane

ATP synthasecristae

ribosome

proton channel

ox ox ox ox

red red red red

34

3. Ubiquinone- Coenzyme Q (CоQ)

2. Flavin Dehydrogenase or Flavoproteins (FP)

They are conjugated enzy-mes with prosthetic groups FMN or FAD (both are active form of vitamin B2).

1. NAD-Dependent Dehydrogenases

It is a small molecule soluble in the lipid part of a membrane due to a non-polar side chain. It can easily move within the membrane. The term "ubiquinone" means the ubiquity of coenzyme Q in nature.Coenzyme Q is a specific collector of electrons and protons from the reduced coenzymes both FMNH2and FADH2. Its reduced form is CoQH2.

NAD+

(oxidized form)

+

NADН(reduced form)

2Н+2е-

Н+

Н

..

They are conjugated enzymes that directly catalyze the oxidation of substrates are called primary dehydrogenases.

NAD+ (the active form of vitamin PP) is a coenzyme of these enzymes. Accepting two protons and two electrons from the substrate it is converted to the reduced form NADH.

FAD

FMN

Н

Н

2Н+2е-

FADН2 (FMNН2)

(reduced)

FAD-containing dehydrogenases catalyze oxidation of different substrates such as succinate, glyceril-3-phosphate, acyl-CoA. In these cases they work as primary dehydrogenases. The dehydrogenasecontaining FMN gains protons and electrons from the reduced coenzyme NADH. It is named NADH-dehydrogenase.

2Н+2е-

ОН

ОН

FAD, (FMN)

(оoxidized)

CоQ

(reduced)

CоQН2

(oxidized)34

35

4. Iron-Sulphur Proteins (FeS)

They are complex proteins containing non-heme iron and sulphur, which can reversibly be oxidized and reduced. Iron-sulphur proteins transfer electrons. They are located in the lipid bilayer of the membrane.These redox systems are stable only within protein molecules. They may contain from 2 to 6 iron atoms forming various complexes with inorganic sulphide and SH-groups of cysteineresidues in proteins. (FeS) proteins are associated with FAD, FMN and cytochrome b.

All electron transport chain participants are structurally organized into four redox systems -multienzyme complexes (I - IV), embedded in the lipid matrix of the inner mitochondrial membrane.The oxidation process begins with the transfer of electrons and protons (2H+ 2e) from the oxidized substrate to NAD+ or FAD ( it depends on the substrate nature).

protein

5. Cytochromes

protein

heme

These are conjugated proteins containing heme(hemoproteins) similar to hemoglobin heme which is tightly bound prosthetic group. Heme contains hemin iron that can change its valence by transfer only electrons:

Cytochromes differ from each other by protein components and heme substituents. It results in differences of their physical, chemical properties and redox-potentials.

Cytochrome aa3, or cytochrome oxidase, is a terminal enzyme that transfers electrons directly to oxygen (aerobic enzyme). It consists of six subunits apart from heme iron. Also cytochrome oxidase contains copper atoms which transport electrons. Initially iron atoms of cytochrome aa3 take part in the electron transfer, and then copper atom of aa3.

Cytochromes are electron carriers in the respiratory chain. They are arranged according to the values of their redox potentials in the following way: cyt.b, cyt.c1, cyt.c, cyt.a, cyt.a3.

Molecular Organization of the Electron Transport Chain

SH2

36

Complex I , or NADH dehydrogenase, is a flavoprotein containing FMN. This enzyme oxidizes NADH and transports two hydrogen atoms (2H+ 2e-) to coenzyme Q. This complex also includes (FeS) proteins.

Complex III, or ubiquinone-dehydrogenase, is the enzyme complex consisting of cytochrome b, (FeS)-protein and cytochrome c1. This complex transports electrons 2e-

from the reduced ubiquinone CoQH2 to cytochrome c. Cytochrome c is a small water-soluble protein located on the outer surface of the inner mitochondrial membrane.

NADН NAD+

succinate

acyl-CоА

membraneGlycerol-3-phosphate

mitochondrial

matrix

SH2isocitratepyruvatemalate

glutamate

There are another FAD-dependent dehydro-genases in the mitochondrial matrix. They oxidize the appropriate substrates (glycerol-3-phosphate, acyl-CoA) and transfer hydrogen atoms to coenzyme Q.

Complex II, or succinate dehydrogenase, is flavoprotein containing FAD. This enzyme oxidizes succinate and transports two hydrogen atoms (2H+ 2e-) to coenzyme Q. The complex includes (FeS) proteins.

Coenzyme Q is the final component of the respiratory chain capable for transfer both protons and electrons (2H+ 2e-). Later protons (2H+) are transferred from the inner surface of the mitochondrial membrane to the outer one, while the electrons (e-) are transferred onto oxygen through the chain of cytochromes.

Complex IV, or cytochrome c oxidase, is the enzyme complex consisting of cytochromes a and a3. Complex IV carries out the final stage of the biological oxidation. This stage includes thereduction of oxygen into water by 2H+ and 2e-:

2е + ½ О2 О2-

Oxygen binds free protons 2H+ in the mitochondrial matrix, resulting in water formation:

2Н+ + ½ О2- Н2О

Currents of hydrogen atoms are fused at the stage of the reduced CoQH2.

fumaratesuccinateNADHН NAD+ ½ О2+2Н Н2Оmаtrix

intermembrane

space

Q

III

Fe-S

37

Redox potential of the pair NAD+/NADH is - 0,32 V, which indicates a high capacity of donating electrons. Redox potential of the pair oxygen/water is 0,82 V, which indicates high ability to gain electrons.

Passage of electrons through the mitochondrial ETC, or the mitochondrial respiratory chain, results in energy release. Most of this energy (60%) is dissipated as a heat. The rest is accumulated in the high-energy bonds of ATP molecules that are produced by oxidative phosphorylation.

The order of electron carriers in the mitochondrial respiratory chain depends on the magni-tudes of their redox-potentials. To provide spontaneous transfer of electrons the compounds of the ETC are arranged according the gradient of their redox-potentials:from minimal to maximal magnitude.

Heat ADP + P АТP

О2 Н2ОElectron Transport Chain (ETC)

-0,32

+0,82

Total difference of redox- potentials magnitudes is 1,14 V. It corresponds to the change in free energy ∆G = -220 kJ / mol. The total amount of energy of the reaction is divided into small portions. Their magnitudes depend on the difference between the redox-potentials of the corresponding redox-pairs. It is assumed that this division into portions provides a surprisingly high energy yield of the respiratory chain - approximately 60%.

Complex I

FMN FeSComplex III

b FeS c1

Complex IV

a a3сQ

SH2

NAD+FAD FeS

Complex II

SH2

1/2О2

Н2О

2Н

Energy

38

This phosphorylation is called oxidative because energy for the formation of high-energy bonds is generated in the oxidation process due to the movement of protons and electrons along the mitochondrial ETC.

The main electron carriers are complexes I, III, IV. They use energy of electron transport and provide transfer of protons from the matrix into the intermembrane space. As a result, pH value is reduced and electrochemical potential ∆µH+ appears (chemiosmotic theory by P.Mitchell). This potential is a driving force for synthesis of ATP from ADP and phosphoric acid. ATP synthesis is associated with the reverse flow of protons H+ from the intermemb-rane space into the matrix. The inner mitochondrial membrane is impermeable for protons. Reverse flow of protons according to the gradient of their concentration occurs due to a special enzyme called ATPase or ATP-synthase (complex V). When a certain value of ∆µH+ is reached, ATPase is activated.There is a channel through which protons return back into the matrix according to the gradient of their concentration. This proton channel is the compound of ATP-synthase. The energy of ∆µH+ is used for ATP synthesis.

matrix

intermembrane

space

NADНН NAD+

succinatefumarate

ATP synthesis

Chemical potential ∆рН Electrochemical

potential ∆µН+

½ О2+2Н+ Н2О

ADP+P

ATP-synthase

V

Mechanism of Oxidative Phosphorylation

cyt.сН+

Н+Н+

Н+

39

Substrates oxidized by NAD-dependent dehydrogenases (pyruvate, ά-ketoglutarate, isocitrate, malate, glutamate, lactate) are energetically more valuable because their oxidation produces three ATP molecules (P/O = 3). The oxidation of the substrates by FAD-dependent dehydrogenases (succinate, acyl-CoA, glycerol-3-phosphate) generates 2 ATP molecules (P/O = 2) .

Each of three complexes (I, III, IV) provides a proton gradient necessary for ATP-synthaseand synthesis of 1 ATP molecule per each.

NAD+

SH2 (P/O = 3)Pyruvate

α-KetoglutarateIsocitrateMalate

GlutamateLactate

SH2 (P/O=2) Succinate

Acyl-CoA

Glycerol-3-phosphate

О2

Complex I

FMN FeSComplex III

b FeS c1

Complex IV

a a3сQ

АDP + PАТP

АDP + PАТP

ATP + PАТP

FAD FeS Ratio of oxidative phosphorylation

(P/O) means the number of phosphoric acid molecules (P) used in ATP synthesis for every oxygen atom (O) consumed.

P/O magnitude depends on chemical structure of the oxidized substrates SH2 .

The ratio ATP/ADP is named respiratory control. Respiratory control coordinates the rate of all catabolic pathways and the rate of tissue respiration. The decrease of ATP /ADP ratio stimulates the oxygen consumption and leads to the activation of tissue respiration and oxidative phosphorylation.

The processes of the reducing equivalents (protons and electrons) transfer through the mitochondrial ETC and ATP synthesis (tissue respiration and oxidative phospho-rylation) are tightly coupled. They occur only at the same time and their rates change simultaneously.

Complex II

40

Inhibitors block the respiratory chain by binding to the specific enzymes or coenzymes in the certain sites of the ETC. In this case the P/O is decreased even to zero depending on the site of the inhibitor action:

a) rotenone (an insecticide), barbiturates (sleeping medicine);

b) antimycin A(an antibiotic);

с) CN-, CO, H2S, azides.

In some cases oxidative phosphorylation and tissue respiration become uncoupled. It leads to the mitochondrial respiration free from ATP synthesis. The uncoupling of tissue respiration and oxidative phosphorylation may be caused both by damage of the mitochond-rial membranes and by action of substances that are named uncoupling agents. These lipophilic substances such as 2,4-dinitrophe-nol, fatty acids (FA) transport protons across the inner mitochondrial membrane avoiding ATPase and thus destroying the proton gradient. Protein thermogenin is the natural uncoupler. It works only as a proton channel in the mitochondria of brown adipose tissue. The end result of the any uncoupler action is a decrease of P/O ratio and dissipation of energy as a heat.

The Inhibitors of Electron Transport and Oxidation Phosphorylation

Antibiotic oligomycin is the inhibitor of ATP synthesis in the mitochondrial ETC by oxidative phosphorylation. It inhibits the activity of ATP-synthase.

1. Membrane damage

2. Mobilecarriers

3. Controlledproton channel

fats FA

norepinephrine

thermo-genin

Rotenone, barbiturates

Antimycin A

СN- or СО

а)

в)

с)

NADН CоQ cyt.b cyt.с1 cyt.с cyt.аа3 О2

NADН CоQ cyt.b cyt.с1 cyt.с cyt.аа3

NADН CоQ cyt.b cyt.с1 cyt.с cyt.аа3 О2

О2

41

COMMON PATHWAYS OF CATABOLISM1. Oxidative Decarboxylation of Pyruvate

Pyruvate is oxidatively decarboxylated to acetyl-CoA to enter the citric acid cycle or to be used for fatty acids synthesis. The oxidative decarboxylation of pyruvate is catalyzed by multienzyme complex that is called pyruvate dehydrogenase comlpex (PDH).

thiamin pyrophosphate (TPP) lipоamide (LA)

FADNAD+cоenzyme А (HS-CоА)

It consists of three enzymes (Е1 , Е2 , Е3 ) and five cofactors:

Pyruvate +NAD+ + НS-CоА СО2+Acetyl-CoA+NADH+Н+

О

Е1 –ТPP (pyruvate dehydrogenase) catalyzes decarboxylation of pyruvate to produce hydroxyethyl-TPP .

Е2 –LA (dihydrolipoyl acetyltransferase) brings about the formation of acetyl lipoamide from hydroxyethyl-TPP and then catalyzes the transfer of acetyl residue to produce acetyl-CoA. During this stage disulphide bond of oxidized lipoamide is reduced leading to the formation of dihydrolipoamide.

Е3-FAD (dihydrolipoyl dehydrogenase) catalyzes the convertion of reduced lipoamide(dihydrolipoamide) to oxidized lipoamide, transferring the reducing equivalents to FAD. The reduced FADH2 transfers protons and electrons to NAD+ to yield NАDН Н+, which can pass through the mitochondrial respiratory chain to give 3 ATP molecules.

Prosthetic groups: they are linked to apoenzymes by covalent bonds and can not be dissociated from

the apoenzymes

Coenzymes: they are linked to apoenzymes by weak noncovalent bonds and are easily

dissociated from the apoenzymes

Е1 –ТPP pyruvate

dehydrogenase

Е2 –LAdihydrolipoyl

acetyltransferase Е3-FAD dihydrolipoyldehydrogenase

TPP

TPP

Pyruvate

Acetyl-CoACoA CoA

FAD

FAD H2

LA

NAD+

NАDН Н+

42

2. THE CITRIC ACID CYCLE

The citric acid cycle (Krebs cycle, tricarboxylic acid cycle) is a series of reactions in mitochondria resulting in the oxidation of acetyl-CоА (СН3СО∼SCоА) to carbon dioxide СО2 and liberation of hydrogen atoms (Н) to reduce NAD and FAD in the mitochondrial respiratory chain for the release of energy of biological molecules. Citric acid cycle (CAC) acts as the final common way for the oxidation of carbohydrates, lipids and proteins, because all these substances are cleaved to acetyl-CoA or to the intermediates of the CAC. Besides this, CAC is the source of the precursors for gluconeogenesis, fatty acids and heme synthesis. The CAC intermediates take part in transamination and deamination. Therefore CAC plays an important role both in catabolic and anabolic pathways, that is why it is considered to be an amphibolic pathway.

Metabolites and enzymes of CAC are located in mitochondrial matrix and in the inner mitochondrial membrane. They are associated with the mitochondrial respiratory chain. It allows the reducing equivalents (4 pairs of hydrogen atoms Н) that are produced during oxidation of the CAC metabolites to enter the mitochondrial respiratory chain by means of primary hydrogen atoms acceptors NАDН+Н+ and FАDН2.

Hydrogen atoms are passed down a redox gradient of carriers to their final reaction with oxygen (О2 ) to form water Н2О. Ion transport in the mitochondrial respiratory chain is linked with ATP production by oxidative phosphorylation.

Acetyl-CоА is considered to be the «аctive acetic acid» (it has 2 carbon atoms - С2). Acetyl-CoA is a product of the oxidative decarboxylation of pyruvate. In the first reaction of CAC acetyl-CoA is condensed with oxaloacetate containing 4 carbon atoms (С4). The reaction yields citrate – 6-carbon atoms substance (С6). In the following reactions of CAC 2 carbon dioxide molecules are liberated (2 СО2). All further reactions of the CAC are aimed at the oxaloacetate (С4) regeneration. The overall equation for CAC is:

СН3СООН + 2Н2О 2СО2 + 8Н

СО2

СО2

(С2)

(С4)

(С6)

(С6)

(С5)

(С4)

(С4)

(С4)

(С4)

citrate

isocitrate

α-ketoglutarate

succinyl∼SCоА

succinate

fumarate

malate

oxaloacetate

Acetyl-CоА

NADНNADН

NADН

FADН2

FAD

CACNAD+

NAD+

NAD+

GTPATP

43

∼SКоА

∼SКоА

CAC

НSКоА

citrate

cis-aconitate

isocitrate

α-ketoglutarate

succinyl∼SКоА

fumarate

malate

succinate

oxaloacetate

НSCоА

dehydration

condensation

1

2а

rehydration

2в

GTP

окислительноедекарбокси-лирование

oxidativedecarboxylation

4

decarboxy-

lation

3

substrate level phosphorylation

5

НSCоА

GDP+P

dehydrogenation

6

dehydrogenation

8

hydration

7

NADНН+

FADН2

cytrate synthase

aconitase

aconitase

isocitratedehydrogenase

succinatedehydrogenase

α-keto-glutarate

dehydrogenase

malatedehydrogenase

fumarase

СО2

СО2succinatethiokinase

3

44

REGULATION OF THE CITRIC ACID CYCLE

Acetyl-CоА

citrate synthase citrate

isocitrate

α-keto-glutarate

dehydrogenase

isocitratedehydrogenase

α-ketoglutarate

succinyl∼SCоА

GTP

succinatedehydrogenase

malate

malatedehydrogenase

oxaloacetate

Pyruvate

pyruvatedehydrogenase

complex

АTP, acetyl-CоА, NADН, fatty acids

АMP, НSCоА, NAD+, Са2+

АТP, NАDН, succinyl-CоА

АDP

АTP

АDP, Са2+

NADН, succinyl-CоА

Са2+

NADН

FADН2

CAC

The most important regulatory mechanism of the citric acid cycle isNАDН/NАD+ ratio.

activation

inhibition

GENERATION OF HIGH ENERGY PHOSPHATE IN THE CAC

The oxidation of one molecule of acetyl-CoA in CAC results in production of reduced coenzymes: 3 molecules of NADН+Н+ and 1 molecule of FADН2. They are oxidized in the mitochondrial respiratory chain generating 11 ATP molecules: 3 ATP molecules for each NADН+Н+ and 2 ATP for FADН2 (3х3 + 2х1 = 11). The mechanism is oxidativephosphorylation. Besides this, 1 ATP molecule is produced in CAC itself by substrate level phosphorylation. In this reaction the energy of succinyl∼SCоА is used to yield GTP from GDP and inorganic phosphate with the following production of ATP from ADP and GTP.Thus, the net quantity of ATP molecules formed in CAC is 12 (11 + 1 = 12) .

succinate

fumarate

45

Multiple Choice QuestionsTest 1The citric acid cycle acts as the final common pathway for the oxidation of carbohydrates, lipids and proteins. Which reaction does it start with? А.Decarboxylation of α-ketoglutarateВ.Dehydrogenation of isocitrateС.Condensation of acetyl-CoA with oxaloacetateD.Convertion of malate to oxaloacetateE.Isomerization of citrateTest 212 ATP molecules are formed per one turn of the citric acid cycle (CAC). Most of them are generated in the mitochondrial respiratory chain. Which substances produced in the CAC are donors of the reducing equivalents for it?A.NADPH2 and NADH2B.NADH2 and THFAC.NADH2 and FMNH2D.THFA and FADH2E.NADH2 and FADH2Test 3The citric acid cycle (CAC) plays very important role in metabolism. Choose the correct statement about it.A.All reactions of the CAC are located in cytosolB.The CAC is amphibolic pathwayC.The only mechanism of ATP generation in the CAC is oxidative phosphorylationD.Vitamins B12 and folic acid play key role in the CACE.One molecule of CO2 is produced in the CACTest 4Oxidative decarboxylation of pyruvate needs three enzymes and five coenzymes. Which enzyme of this metabolic pathway is the regulatory one?A.Dihydrolipoyl dehydrogenaseB.Pyruvate carboxylaseC.Dihydrolipoyl transacetylaseD.Pyruvate dehydrogenaseE.Pyruvate kinaseTest 5Oxidative decarboxylation of pyruvate is catalyzed by the pyruvate dehydrogenasemultienzyme complex. What is its end product?A.Acyl-CoAB.FADH2C.Acetyl lipoamideD.Acetyl-CoAE.Oxaloacetate

KEYS: 1 – C; 2 – E; 3 – B; 4 – D; 5 – D.

46

Carbohydrates are the most abundant among the major classes of biomolecules. Chemically, they are simple organic compounds that are aldehydes or ketones with many hydroxyl groups added usually to each carbon atom not part of the aldehyde or ketonefunctional group.

METABOLISM OF CARBOHYDRATES

Functions of Carbohydrates:storage and transport of energy (monosaccharides, homopolysaccharides)structural components (heteropolysaccharides, glycoproteins, DNA, RNA)

aldose ketoseR R

glucose galactose fructose

α-1,6 glycosidicbonds

α-1,4 glycosidic bonds

Glycogen: it is very similar to amylopectin, the branched polyglucose molecule found in starch. The only difference is that glycogen is more highly branched.

Polysaccharides:1. homo-

starch (amylose and amylopectin)glycogen cellulose

2. hetero-

Disaccharides:maltose sucroselactose

Monosaccharides:1. hexoses (6C):

glucose (aldose)fructose (ketose)galactose

2. pentoses (5 C)3. trioses (3C)

Starch is a branched homopolysaccharide, consists of glucose monomers, contains α-1,4-and α-1,6 glycosidic bonds

47

4. Dichotomic degradation of glucose (indirect oxidation of glucose):4.1. Anaerobic glycolysis is glucose oxidation in anaerobic conditions resulting in

formation of 2 lactate molecules and 2 ATP molecules.4.2. Aerobic glycolysis is glucose oxidation in aerobic conditions resulting in formation 2

pyruvate molecules and 8 ATP molecules.4.3. Alcoholic fermentation is glucose convertion to ethanol caused by microorganisms.

5. Gluconeogenesis is glucose synthesis from noncarbohydrates (amino acids, lactate, pyruvate, the CAC intermediates).6. Apotomic way of carbohydrate metabolism, or direct oxidation of glucose, or

pentose monophosphate pathway

Major Pathways of Carbohydrate Metabolism 1. Digestion of carbohydrates2. Glycogen synthesis (glycogenesis)3. Glycogen degradation ( glycogenolysis)

7. Catabolism of sugars other than glucose

Food carbohydrates

glucose

glyceraldehyde-3-phosphate

glycogen

pyruvate lactate

CAC

ATP

СО2

Н2О

acetyl-CоА

СО2

proteins

ethanol

lipids glycerol

amino acids

ribose-5-phosphate

NADPН+

fructose galactose

nucleotidescoenzymes

DNA, RNAlipids

glucose -6-phosphate

electron transport chain

СО2

48

1. Digestion of Carbohydrates

Digestion of carbohydrates is the hydrolysis of glycosidic bonds in poly- and oligosaccharides carried out by a group of enzymes called glycosidases (the hydrolases).

Maltose

Sucrose

Lactose

glucose + glucose +

glucose + fructose

glucose + galactose

+

+

maltase

sucrase

lactase

pancreatic α-amylase

Oral cavity

salivary α-amylase

α-1,6-glucosidase

Carbohydrates are nutrients for which the digestion begins in the oral cavity by salivary α-amylase (рН opt = 6.7). This enzyme acts on starch randomly and cleaves α-1,4 glycosidicbonds. α-amylase is a an endoenzyme which hydrolyzes inner bods. The end products of carbohydrates digestion in the mouth are oligosaccharides (dextrins) and disaccharides (maltose).

In the small intestine pancreatic α-amylase attacks α-1,4 glycosidic bonds. The products are disaccharides and oligosaccharides. The enzyme α-1,6 glycosidase cleaves α-1,6glycosidic bonds. Disaccharidases are capable for hydrolysis of disaccharides (maltase, sucrase and lactase):

In the duodenum:

In the stomach (рН 1,5-2) carbohydrates are not

digested.

49

Metabolism of Glucose in Tissues

glucose glucose-6-phosphate

ATP ADP

hexokinaseglucokinase

2. Glycogen Synthesis, or Glycogenesis

Hexokinase (all tissues) and glucokinase (liver) carry out the first reaction. This type of reaction - the transfer of the terminal phosphate group from ATP. Glucokinase is specific to glucose and it works in case of high level of glucose (КM = 12 mM). Hexokinasephosphorylates differenthexoses. It is inhibited by glucose-6-phosphate (КM = 0.1 mM).

The cell membranes are impermeable to glucose-6-phosphate

glucose-6-phosphate glucose-1-phosphate

phosphoglucomutase

UTP PP

glucose-1-phosphate uridylyltransferase

UDP-glucose

Glycogen is a polymeric form of glucose storage in the liver and muscles. It is very similarto amylopectin, the branched polyglucose molecule (glucose residues are linked withα(1→4)-glycosidic bonds) found in starch, the only difference is that glycogen is more branched.

Why do organisms store glucose in polymeric rather than in free form? Free glucose would cause an inacceptablyhigh osmotic pressure inside the cell. This means that the osmotic pressure is proportional to the number of molecules per volume, or the molar concentration. Polymerization of glucose vastly reduces its molar concentration and therefore its osmotic activity; it makes storage of large amounts of glucose ‘bio-compatible’.

Granules of glycogen

1) glucose activation

The following steps occur in glycogen synthesis: 1. Glucose activation2. Initiation of glycogen synthesis

3. Chain elongation 4. Introduction of branch points

consists of the formation of UDP-glucose from glucose-6-phosphate, which is converted to glucose-1-phosphate by the enzyme phosphoglucomutase. Glucose-1-phosphate is then activated to UDP-glucose by glucose-1-phosphate uridylyltransferase; this reaction uses uridine triphosphate (UTP).

50

glycogen synthase

branching enzyme

UDP-glucose

glycogensynthase

UDP

glycogen consisting of (n+1) glucose molecules

Oligosaccharide, the primer of glycogen, consists of n glucose molecules (n>4)

uracil

Glycosidic bonds in the linear polysaccharide are in the - α1-4 configuration.

3) elongation

4) introduction of branch

2-3. Initiation and ElongationThe first glucose molecule is attached to the OH group of tyrosine residue in the small protein glycogenin, which therefore serves as a seed for glycogen synthesis. Subsequently, another glucose subunit is attached to the 4-OH group of the first one, and this process is then repeated over and over, giving rise to a long, linear polysaccharide. UDP-glucose is the substrate in both the initiation step and the repetitive chain elongation steps. At this point, all glycosidic bonds in the polysaccharide are in the - α1-4 configuration, so that this stage of nascent glycogen resembles amylose. The enzyme responsible for the initiation and extension of the linear polymer is glycogen synthase.

4. Introduction of Branch PointsBranching enzyme cuts a string of glucose residues from the growing end and grafts it onto the sixth C atom of the glucose residue within the chain. Branching can be repeated, so that thedistance between the adjacent branch points will be 6-8 glucose residues, and branches will carry other branches in their turn.

What is the purpose of this?Branching increases the number of free ends for attachment or removal of single glucose residues during degradation .

51

3. Glycogen Degradation

glycogen ( n molecules of glucose)Н3РО4

glycogen ( n-1 molecules of glucose)

glycogenphosphorylase

glucose-1-phosphate

glycogen

α-1,6- glycosidic bond

glycogenphosphorylase

glucose-1-phosphate

debranchingenzyme

debranchingenzyme

glucose

the lineal (α1,4-) polymer –substrate for

glycogen phosphorylase

activity of glucosyltransferase

activity of 1,6-glucosidase

Glycogen phosphorylase cleaves α- 1,4-glycosidic bonds by means of phosphoric acid, until 4 residues of glucose remain before the branching point of this chain.

Debranching enzyme transfers the remaining fragment of the molecule to another free end. However, it leaves behind a single glucose residue attached by α-1,6-glycosidic bond, which it subsequently cleaves by hydrolysis and releases as glucose. This enzyme has both glucosyltransferase and glucosidase activities.•Transferase: it transfers three glucose residues from the four-residue glycogen branch to a nearby branch.•Glucosidase (amylo-α-1,6-glucosidase): itcleaves the remaining α-1,6 linkage, producing glucose and a linear chain of glycogen which undergoes glycogen phosphorylase action.

Two enzymes collaborate in glycogen degradation: glycogen phosphorylase and debranching enzyme. The product formed in the first reaction,glucose-1-phosphate, is converted back to glucose-6-phosphate.

In the liver which stores glycogen for the benefit of the entire body, glycogen is dephosphorylated by glucose-6-phosphatase with the following release into bloodstream.

However, muscles certainly use glycogen to a large extent for their own keep-up, and therefore glucose-6-phosphate will often be funneled straight into glycolysis.

52

Regulation of Glycogen Metabolism

inactiveactive

Н2Оprotein phosphatase

glucagon epinephrine

cAMP- dependentprotein kinase

ADPATP

inactive active

protein phosphataseН2О

Glucose-6-P

инсулин

insulin glucagon

1. Allosteric RegulationGlycogen synthase is activated by glucose-6-phosphate, glycogen phosphorylase isinhibited by glucose-6-phosphate, ATP and free glucose in liver2. Hormonal RegulationGlycogen synthesis is increased after a meal when glucose concentration is high. The hormone insulin (pancreas) enhances the phosphodiesterase activity in the liver and lowers cAMP level in cytosol. This hormone activates glycogen synthase by dephosphorylation

Glycogen breakdown is increased when glucose concentration is low. The hormone glucagon that is synthesized in pancreas stimulates the phosphorylation of glycogen phosphorylase by activation of cAMP-dependent proteine kinase. Note that glycogen synthase and glycogen phosphorylase respond to phosphorylation in opposite way: the first enzyme is inactivated, the second one is activated.3. Regulation by Ca++

There are regulatory differences between glycogen phosphorylase in muscles and the liver: glucose inhibits the liver enzyme but not the muscle enzyme, and Ca ++ stimulates the muscle enzyme but not the liver enzyme.

Glycogenesis and glycogenolysis are two opposite proceses, controlled by the enzymes glycogen synthase and glycogen phosphorylase.

Three mechanisms of the enzymes regulation :

Glycogen storage diseasesVon Gierke’s disease means a deficiency of glucose-6-phosphatase. In this disease the release of glucose from glucose-6-phosphate is impaired that leads to low blood glucose level (hypoglycemia). The liver and the kidneys in patients with von Gierke’s disease are enlarged.

cAMP- dependentprotein kinase

glycogensynthase

b

glycogensynthase

a

glycogen phosphorylase

glycogenphosphorylase

53

Glycogen Storage DiseasesGlycogen Storage Diseases

54

Glucose

2 Pyruvate

2 Acetyl-CоА

4СО2 + Н2О

2 Lactate2 Ethanol +2СО2

glycolysis

anaerobic conditions

aerobic conditions

2СО2

anaerobic conditions

in yeast

4. Dichotomic degradation of glucose isindirect oxidation of glucose.

4.1. Anaerobic GlycolysisEmbden-Meyerhof-Parnas pathway

Anaerobic glycolysis occurs mainly in erythrocytes, since mitochondria are absent in them, and in skeletal muscles during strenuous exercise where oxygen supply is limited. Under anaerobic conditions pyruvate is converted into lactate by lactate dehydrogenase (LDH). Accumulation of lactate in the exercising sceletal muscles results in a sensation of pain. The depletion of glucose and ATP leads to gradual exhaustion.In the 1-st phase of glycolysis that needs energy consumption (2 ATP molecules), glucose is phosphorylated, or activated and gradually splitted into two three-carbon compounds:glyceraldehyde-3-phosphate and dihydroxyacetone phosphate.In the 2-nd phase of glycolysis that is chatacteryzed by energy generation, 4 ATP molecules and 2 NADH molecules are produced.

Glycolysis accomplishes the degradation of glucose to pyruvate.Its main purpose is the production of energy (ATP). Glycolysis generates some ATP molecules directly, but a lot more indirectly through the subsequent oxidation of pyruvate. The need of ATP is universal, so that glycolytic pathway is found in every cell of our body.

The ATP generation in anaerobic glycolysis: 2АТP molecules (2х2-2).

In glycolytic reaction catalyzed by glyceraldehyde-3-phosphate dehydrogenase2 molecules of NAD+ are consumed and converted to NADH. Under aerobic conditions NADH is oxidized to NAD+ in the mitochondrial respiratory chain. Under anaerobic conditions NADH is oxidized to NAD+ in the reaction catalyzed by LDH during reduction of pyruvate into lactate.

Glucose

2 Pyruvate 2 Lactate 2 NADНН+

2NAD+

without О2

55

glucose-6-phosphate

ATP

Glucose

fructose-1,6-bisphosphate

ATP

fructose-6-phosphate

(2) 1,3-bisphosphoglycerate

(2) Lactate

2 NAD+2 NADН Н+

2ATP

2ATP

2 NADН Н+

2 NAD+

glyceraldehyde-3-phosphate

dihydroxyacetonephosphate

Reactions 1-3Glucose activation by

phosphorylationare requires 2 ATP

Reaction 4Dichotomy is splitting of

6-carbons molecule into 2 phosphotrioses

Reaction 5the isomerization of

phosphotrioses

Reaction 6Formation of 2 NADНН+ and high-energy

compound

Reaction 7Substrate level

phosphorylation:formation of 2 ATP

Reaction 8 and 9Formation of high-

energy compound and water

Reaction 10Substrate level

phosphorylation:formation of 2 ATP

ener

gy g

ener

atio

n s

tage

en

ergy

-req

uir

ing

stag

eAnaerobic Glycolysis1

2

3

4

5

6

7

8

9

10

11

Reaction 11the hydrogenation

of pyruvate to lactate (NADНН+, formed in reaction 6, is

used)

(2) 3-phosphoglycerate

(2)2-phosphoglycerate

(2) Pyruvate

(2) phosphoenolpyruvate

hexokinase

phosphofructokinase

pyruvate kinase

56

1 - hexokinase2 - phosphohexose isomerase3 – phosphofructokinase4 – aldolase5 – phosphate isomerase

6 – glyceraldehyde-3-phosphate dehydrogenase7 – phosphoglycerate kinase8 – phosphoglycerate mutase9 – enolase10- pyruvate kinase11- lactate dehydrogenase

1 st

age

2 st

age

4. 2. Aerobic Glycolysis

aerobic conditions ETC

Glucose

2 Pyruvate

2NADНН+

2NAD+

Aerobic cleavage of glucose is the major catabolic pathway for glucose in humans. It is the main metabolic process generating energy for brain. In a liver the main function of this metabolic pathway is to produce substrates for synthesis of lipids. Aerobic cleavage of glucose includes the following

stages: 1) aerobic glycolysis that means the gradual convertion of glucose into two molecules of pyruvateunder aerobic conditions; 2) transfer of pyruvate from the cytosol into the mitochondria and oxidative decarboxylation of pyruvate yielding acetyl-CoA;3) oxidation of the acetyl group from acetyl-CoA to carbon dioxide and water in the citric acid cycle (CAC) associated with the mitocondrial electron transport chain (ETC).

СО2 Н2О

acetyl-CоА

Glycolysis is a metabolic pathway that takes place in the cytosol of cells in all living organisms.

Three enzymes namely hexokinase, phosphofructokinase, pyruvatekinase catalysing the irreversible reactions regulate glycolysis .

Crabtree effect means the inhibition of oxygen consumption by the addition of glucose to tissues that are characterized by high aerobic glycolysis. It is opposite to Pasteur effect that means the inhibition of anaerobic glycolysis by oxygen.

57

Glucose

Aerobic and Anaerobic Glycolysis

fructose-1,6-bisphosphate

glyceraldehyde-3-phosphate

dihydroxyacetonephosphate

АТP

Energy consump-tion stage

The phosphorylationof glucose needs 2

ATP

2 NADН Н+

2 NAD+2 NADН

2 NAD+

(2) Lactate(2) Pyruvate

phosphoenolpyruvate

2АТP

2АТP

Н2О

Energy generation stage

1,3-bis-phospho-glycerate

3-phosphoglycerate

4 АТP are formed due to the

substrate levelphosphorylation

2 NADН

2 NAD+(2) Pyruvate

↓30 АТP

2 NАDНН+

are formed under anaerobic conditions. NADHН+ is oxidized into NAD+ by hydrogenation of pyruvate to lactate

Under aerobic conditions NADHН+ is oxidized to NAD+ in the mitochondrial respiratory chain by oxidative phosphorylation: each NADHН+ produces 3 ATP molecules.

2

2

2

1

2

3

45

6

7

8

9

10

CACАТP СО2

Н2О

2аcetyl-SCоА

СО2

АТP

58

In the liver ethanol is oxidized to acetic aldehyde by the enzyme alcohol dehydrogenase. This reaction occurs in both peroxisomes and the smooth endoplasmic reticulum. In the mitochondria the enzyme acetaldehyde dehydrogenase converts acetic aldehyde to nontoxic acetate. Acetate enters the CAC as acetyl-SCoA.

4.3. Alcoholic Fermentation Glucose

Pyruvate

pyruvate decarboxylase

alcohol dehydrogenase

NАDНН+ NАD+

Alcoholic fermentation is the convertion of glucose to ethanol.

EthanolAcetic aldehyde

Pyruvate Lactate

lactate dehydrogenase

NАDНН + NАD +

Metabolism of ethanol by alcohol and aldehyde dehydrogenases involves the consumption of NAD. High concentration of the produced NADH favours the conversion of pyruvate to lactate which may lead to lactic acidosis. Due to the reduced gluconeogenesis hypoglycemia is developed in chronic alcoholism. The CAC is impaired since the availability of both oxaloacetate and NAD is reduced. Acetaldehyde causes headache, nausea, tachycardia and low blood pressure.

Ethanol Metabolism

Acetic aldehydeEthanol

alcohol dehydrogenase

NАD+ NАDНН +

acetaldehyde dehydrogenase

NАD + NАDНН +

Acetate

Acetyl-CoA

СО2

Н2ОАТP

Since the activity of acetaldehyde dehydrogenase is less than that of alcohol dehydrogenase, acetaldehyde is accumulated leading to various complications. Disulfiram (antabuse) that is used for the treatment of alcoholism inhibits acetaldehyde dehydrogenase.

ОН

(ТPP)

59

Glycolysis and Gluconeogenesis

Key enzymes of gluconeogenesis

I – Pyruvate carboxylaseII – Phosphoenolpyruvatecarboxykinase (PEPCK) III – Fructose-1,6-bisphosphataseIV – Glucose-6-phosphatase

Key enzymes of glycolysis

1 – Hexokinase3 - Phosphofructokinase10 – Pyruvate kinase

III

IV

Glucose

glucose-6-phosphate

1

3

fructose-6-phosphate

fructose-1,6-bisphosphate

(2) glyceraldehyde-3-phosphate

Lipids

Glycerol

(2) 3-phosphoglycerate

(2) phosphoenolpyruvate(PЕP)

Proteins

I

II10

(2) 1,3-bisphosphoglycerate

2АТP

2GТP

2АТP

2 NАD+

2 NАDН Н+

(2) oxaloacetate

Aminoacids

(2) Pyruvate

Proteins

Amino acids

(2) Lactate

60

What are the key enzymes in gluconeogenesis?

Pyruvate carboxylase is a mitochondrial enzyme that catalyzes carboxylationreaction converting pyruvate to oxaloacetate using the reaction mechanism involving biotinyl "swinging arm” and ATP hydrolysis. Pyruvate carboxylase is dependent on allosteric activation by acetyl CoA.

Phosphoenolpyruvate carboxykinase(PEPCK) localized in either the mitochondrial matrix or the cytosol (or in both in the case of human liver cells) catalyzes phosphoryl transfer reaction that converts oxaloacetate to phosphoenolpy-ruvate (PEP) using the energy released by decarboxylation and GTP hydrolysis. Transcription of the PEPCK gene is regulated by hormones (cortisol).

5. Gluconeogenesis

The overall reaction of gluconeogenesis2 pyruvate + 2NADH + 4ATP + 2GTP + 6H2O → Glucose + 2NAD+ + 2H+ + 4ADP + 2GDP + 6Pi

Gluconeogenesis produces glucose from noncarbohydrate sources (lactate, amino acids, glycerol and others) mainly in the liver for export to other tissues that need glucose as a source of energy, first of all into the brain and erythrocytes.

61

Key enzymes of gluconeogenesis

Pyruvate carboxylaseacetyl-CоА

Fructose-1,6-biphosphatase

АТP АМP

Glucose-6-phosphatase

Phosphoenolpyruvatecarboxykinase (PEPCK)

glucagonepinephrine

glucocorticoidsinsulin

Key enzymes of glycolysis

Hexokinase Glucose-6-P

Phosphofructokinasecitrate

АТP

Fructose-6-P

АМP

АТP

acetyl-CоА

Pyruvate kinase

Gluconeogenesis, or the conversion to glucose, uses the reversible reactions from glycolysis, and 4 distinct reactions that circumvent the ones from glycolysis that are irreversible. These reactions are catalyzed by pyruvatecarboxylase, phosphoenolpyruvatecarboxykinase, fructose-1,6-bisphosphatase and glucose-6-phosphatase.Gluconeogenesis is essentially confined to two organs: the liver and the kidneys.

Both organs have an ample supply of amino acids: the liver from the intestine via the portal vein, the kidneys because they extract amino acids from the considerable volume (~150 l/day) of plasma ultrafiltrate that represents the first stage of urine secretion.

Gluconeogenesis, or the conversion to glucose, uses the reversible reactions from glycolysis, and 4 distinct reactions that circumvent the ones from glycolysis that are irreversible. These reactions are catalyzed by pyruvatecarboxylase, phosphoenolpyruvatecarboxykinase, fructose-1,6-bisphosphatase and glucose-6-phosphatase.Gluconeogenesis is essentially confined to two organs: the liver and the kidneys.

Both organs have an ample supply of amino acids: the liver from the intestine via the portal vein, the kidneys because they extract amino acids from the considerable volume (~150 l/day) of plasma ultrafiltrate that represents the first stage of urine secretion.

Cori cycle Gluconeogenesis is very important in the utilization of lactate generated in the skeletal muscles. For example, athletes that are exercise intensely for the short periods of time, such as in a sprint race, build up large amounts of lactate in their muscles as the result of anaerobic glycolysis. The “warming down”period of continual movement under aerobic conditions performed by athletes for ~ 15 minutes after a race increases circulation and removes lactate from the muscles. Lactate is transported to the liver where it is converted to glucose by the gluconeo-genic pathway and shipped back to the muscle to replenish glycogen storage.

The combination of glycolysis in peripheral tissues with

gluconeogenesis in the liver is referred to as Cori cycle

62

The most important function of the pentose phosphate pathway is to reduce two molecules of NADP + to NADPH for each glucose-6-phosphate (glucose-6P) that is oxidativelydecarboxylated to ribulose-5-phosphate (ribulose-5P).The pentose phosphate pathway is also responsible for production of ribose-5-phosphate (ribose-5P) from glucose-6P. NAD+ functions as the primary oxidizing agent in the cells (it accepts electrons) whereas NADPH is the primary reducing agent in the cells (it donates electrons).

6. Apotomic Pathway of Carbohydrate Metabolism, or the Direct Oxidation of Glucose, or Pentose Phosphate Pathway (PPP), or the Hexose MonophosphateShunt, or Phosphogluconate Pathway

6. Apotomic Pathway of Carbohydrate Metabolism, or the Direct Oxidation of Glucose, or Pentose Phosphate Pathway (PPP), or the Hexose MonophosphateShunt, or Phosphogluconate Pathway

The pentose phosphate pathway can be divided into two phases:

•the oxidative phase which generates NADPH, and

•the nonoxidative phase which interconvertsC3, C4, C5, C6 and C7 sugar phosphates.

Flux through the oxidative and nonoxidativephases of the pathway is tightly regulated in response to such factors as energy needs of the cell, NADP + /NADPH ratio and the requirements for nucleotide and coenzyme biosynthesis. For example, when NADPH is needed, ribulose-5P is converted back into glucose-6P to maintain flux through the pathway (1), however, if ATP and NADPH are needed (which would be the case for most anabolic pathways), then some of the ribulose-5P is used to synthesis hexosephosphates for glycolysis (2). If the cell needs to increase the rate of nucleotide and coenzyme biosynthesis the most of the ribulose-5P is shunted towards ribose-5P synthesis (3).

63

NADP+

Р

Р

Р

Р

NADP Н

NADP +NADP Н

ННОС

Р

СО ННglucose-6-phosphate

glucose-6-phosphate dehydrogenase

6-phospho-gluconolactone

6-phospho-gluconate

lactonase

6-phosphogluconate dehydrogenase

ribulose-5-phosphate

ribose-5-phosphate

phosphopento-isomerase

1. Oxidative phase 2. Nonoxidative phase(which interconverts C3, C4, C5, C6 and C7

sugar phosphates)

6 molecules of ribulose-5-phosphate

2 moleculesglucose-

6-phosphate

2 moleculesglucose-

6-phosphate

1 moleculeglucose-

6-phosphate

transaldolase

transketolase

Summary PPP:

«apex»

2С5xylulose- 5-phosphate

2 С5xylulose-5 -phosphate

2 С5ribose-5-phosphate

2С3glycer-aldehyde-3-phosphate

2С3glycer-

aldehyde-3-phosphate

2 С7sedoheptulose -

7- phosphate

2С4erythrose -4-phosphate

2С6fructose-6-phosphate

2С6fructose-6-phosphate

is feedback-inhibited by NADPH. Defects in glucose-6P dehydrogenase caused by beans Vicia faba in diet is called favism.

6 Glucose-6P + 12 NADP+ + 12 H2O --> 5 Glucose-6P + 12 NADPH + 12 H+ + 6 CO2

6 Glucose-6P + 12 NADP+ + 12 H2O --> 5 Glucose-6P + 12 NADPH + 12 H+ + 6 CO2

transketolase

64

Three enzymes are required for the oxidative phase: 1.Glucose-6-phosphate dehydrogenase reduces one molecule of NADP+ to NADPHand produces 6-phosphogluconolactone.2. Gluconolactonase cleaves the internal ester bond and produces 6-phosphogluconate.3. 6-Phosphogluconate dehydrogenase reduces another molecule of NADP+ and decarboxylates 6-phosphogluconate to the pentose ribulose-5-phosphate.After completion of the oxidative phase, NADPH generation is over, and everything that remains is juggling sugars in order to regenerate hexoses from pentoses.

The reactions of the second phase:1.Two molecules of ribulose-5-phosphate areconverted to xylulose-5- phosphate and toribose-5-phosphate by ribulose-5-phosphateepimerase and ribulose-5-phosphateisomerase, respectively.

2. Transketolase transfers a C2 unit from the xylulose-5-phosphate to the ribose-5-phosphate, yielding glyceraldehyde-3-phosphate and the C7 sugar sedoheptulose-7-phosphate.

3. Transaldolase transfers a C3 unit from the sedoheptulose-7-phosphate back to glyceraldehyde-3-phosphate, yielding fructose-6-phosphate and the C4 sugar erythrose-4-phosphate. The fructose-6-phosphate may enter glycolysis, or may be converted back to glucose-6-phosphate by phosphohexose isomerase.

4. Transketolase transfers a C2 unit from another molecule of xylulose-5-phosphate to erythrose-4-phosphate.This yields a second molecule of fructose-6-phosphate and again glyceraldehyde-3-phosphate. Both may enter glycolysis or may be converted back to glucose-6-phosphate.

Transketolase employs the coenzyme thiamine pyrophosphate (TPP).

65

Not all cells that require large amounts of NADPH have high activity of the PPP. Some alternative mechanisms that provide NADPH production are the following:

1. Oxidative decarboxylation of malate in the cytosol catalyzed by NADP-dependent malic enzyme.

2. Oxidative decarboxylation of isocitrate to α-ketoglutarate catalyzed by the cytosolic NADP+-dependent isocitrate dehydrogenase which is also located in mitochondria like the CAC enzyme NAD-dependent isocitrate dehydrogenase. NADPH molecules produced in the above reactions are important for fatty acids synthesis in a liver and in adipose tissue.

Cytochrome P450 enzymes split molecular oxygen (O2) and use NADPH to reduce one oxygen atom into water. Another oxygen atom is incorporated into different hydrophobic substrates. For example, this enzymes are used in hydroxylation reactions for synthesis of steroids. Cytochrome P-450 enzymes also participate in the detoxication of hydrophobic drugs by hydroxylation of aliphatic and aromatic moieties in these molecules. The introduced hydroxyl group is used in the following conjugation reactions that occur before the excretion of the modified substance into the urine.

Alternative Sources of NADPH

Uses of NADPH:1.Synthesis of fatty acids.2. Synthesis of cholesterol.3. Fixation of ammonia by glutamate dehydrogenase.4. Oxidative metabolism by cytochrome P-450 enzymes:(a) generation of catecholamine transmitters (dopamine, epinephrine and norepinephrine);(b) drug metabolism.5. Generation of nitric oxide and reactive oxygen species by phagocytes.6. Scavenging of reactive oxygen species that are formed as byproducts of oxygen transport through the mitochondrial respiratory chain.

Uses of NADPH:1.Synthesis of fatty acids.2. Synthesis of cholesterol.3. Fixation of ammonia by glutamate dehydrogenase.4. Oxidative metabolism by cytochrome P-450 enzymes:(a) generation of catecholamine transmitters (dopamine, epinephrine and norepinephrine);(b) drug metabolism.5. Generation of nitric oxide and reactive oxygen species by phagocytes.6. Scavenging of reactive oxygen species that are formed as byproducts of oxygen transport through the mitochondrial respiratory chain.

66

The pentose phosphate pathway is also responsible formaintaining high levels of NADPH in red blood cells (erythrocytes) for use as a reductant

in the glutathione reductase reaction

Glutathione (GSH) is a tripeptide

(γ-glutamylcysteinylglycine) that has a free thioalcohol (-SH) group which functions as an electron donor in a variety of coupled oxidation-reduction reactions in the cells. In erythrocytes, electrons from glutathione are used to keep cysteine residues in hemoglobin in the reduced state, and for reducing harmful reactive oxygen species and hydroxyl free radicals that damage proteins and lipids through oxidation induced cleavage reactions. When erythrocytes are exposed to chemicalsthat generate high levels of superoxide radicals, GSH is required to reduce these damaging compounds. During its function glutathione is oxidized with the following formation of disulphide linkage between two molecules of the oxidized glutathione: GSSH. The enzyme glutathione reductase is used to regenerate GSSH into two molecules of GSH by means of NADPH. An active pentose phosphate pathway in erythrocytes normally provides sufficient levels of NADPH to maintain the GSH:GSSG ratio at about 500:1. Glucose-6P dehydrogenase (G6PD) deficiencyis the most common enzyme deficiency in the world, effecting over 400 million people.

Antimalarial drug primaquine induces acute hemolytic anemia (red blood cell lysis).People with glucose-6-phosphate dehydrogenase deficiency cannot tolerate primaquinebecause their erythrocytes do not contain enough GSH to detoxify the reactive oxygen species produced by the compound. In fact, the reason primaquine works as an anti-malarial drug is because productive infection of the mosquito-borne microorganism Plasmodium is inhibited in erythrocytes under conditions in which NADPH levels are reduced due to increased oxidative stress.

67

7. Catabolism of Sugars Other than Glucose

glucose

fructose

Sucrose

Lactose

Glycogen

Maltose

maltase

sucrase

lactase

glucose-6-phosphate

fructose-6-phosphate

fructose-1,6-bisphosphate

glyceraldehyde-3-phosphate

hexokinase

phosphorylase

hexokinase

fructose-1-phosphate

phosphofructokinase

glyceraldehyde dihydroxyacetonephosphate

АТP

АТP

АТP

АТP

+

Р

fructose-1-phosphate-aldolase

Galactose

UDP - galactose

Fructose intolerance is a hereditary disease that is due to a homozygous defect in the aldolase B gene. In this condition, fructose is still phosphorylated by fructokinase. The resulting fructose-1-phosphate, however, cannot be processed further, and therefore the phosphate tied up in it cannot be reclaimed. Since phosphate is required for the regeneration of ATP from ADP, this means that ATP will be lacking, too, which will sooner or later destroy the cell. The disease is characterized by potentially severe liver failure.A defect in the gene encoding fructokinase leads to a condition named fructosemia or fructosuria. As these names suggest, fructose levels are increased in blood and urine. In this condition, no phosphate depletion occurs, and the liver cells do not incur any damage.

galactose-1-phosphate

galactokinase

АТP

glucose-1-phosphate

UDP-glucose

galactose-1-phosphateuridyltransferase

Classical galactosemia is due to deficiencyof enzyme galactose-1-phosphate uridyltransferase, leading to increased galactose level in circulation. The accumulated galactose is diverted for the production of galactitol by the enzyme aldose reductase. Galactitol (like sorbitol) has been implicated in the development the cataract.

pyruvate СО2 Н2О+

Fructosuria is a rare harmless asymptomatic condition caused by lack of the liver enzyme called fructokinasewhich is needed to turn fructose into glycogen. Symptoms: sugar in urine, presence of fructose in blood.

Lactose Intolerance In lactose intolerance, lactase is missing, and lactose goes down the pipe into the large intestine, where bacteria cleave it to hydrogen gas and other metabolites. Exhaled hydrogen can be used as a diagnostic marker for lactose intolerance.

68

Multiple Choice Questions

Test 1Galactosemia has been revealed in a child. What enzyme deficiency causes this disease?A. Galactose-1-phosphate uridyl transferaseB. Amylo-1,6-glucosidaseC. PhosphoglucomutaseD. GalactokinaseE. Hexokinase

Test 2One of the glycogenoses is characterized by diminished exercise tolerance, high glycogen content in muscles and little concentration of lactate in blood after exercise. This disease is caused by congenital deficiency of the enzyme: A. Glycogen synthaseB. Gamma-amylase C. Alpha-amylase D. Glucose-6-phosphate dehydrogenaseE. Glycogen phosphorylaseTest 3Aspirin was prescribed to a 3-year-old child with fever. It resulted in the erythrocytehaemolysis in a patient. Hemolytic anemia may be caused by congenital insufficiency of the enzyme:

A. Glucose- 6-phosphatase B. Glucose-6-phosphate dehydrogenaseC. Glycogen phosphorylaseD. Glycerol phosphate dehydrogenaseE. Gamma-glutamyl transferase

Test 4Buffer capacity of blood was decreased in the worker due to the exhausting physical labour. This

change may be caused by the increased concentration of ... in his blood.A. Lactate B. PyruvateC. 1,3-Bisphosphoglycerate D. α-KetoglutarateE. 3-Phosphoglycerate

Test 5The myocyte cytosol contains a variety of dissolved metabolites of glucose oxidation. Name one of them that is converted into lactate directly.A. PyruvateB. OxaloacetateC. GlycerophosphateD. Glucose 6-phosphateE. Fructose 6-phosphate

KEYS: 1 – A; 2 – E; 3 – B; 4 – A; 5 – A.

69

Lipids are hydrophobic and various by their chemical structure organic substances.

METABOLISM OF LIPIDS

Basic Lipid Classes

1. Neutral fats - triacylglycerols (TAG) are esters of glycerol bound tofatty acids (FA)

Free fatty acids (FFA) are found in the most human lipids. Like gluco-se they are the most important "fuel molecules" and the sources of energy due to the large number of hydrogen atoms included in them. For example:

RСООН

Saturated fatty acidsС15Н31СООН palmiticС17Н35СООН stearinic

Unsaturated fatty acids:С17Н33СООН oleinic

С17Н31СООН linoleic

С17Н29СООН linolenic

С19Н31СООН arachidonic

Free fatty acids (FFA) areamphipathic substances, as they have hydrophilic(charged) "head"and hydrophobic (uncharged)"tail":

Saturated Fatty Acid

UnsaturatedFatty Acid

Hydrophobic“tail”

Hydrophilic “head”

1.Triacylglycerols are the most compact and capacious form of energy storage. They are hydrophobic substances, so they form structure isolated from water.

Saturated FA

Unsaturated FA

Saturated FA

3. Steroids are diphilic substances:

cholesterol;steroid hormones;bile acids;vitamin D.

Hydrophilicpart

Hydrophobicpart

Glycerophospholipids are diphilic substances,that allows them to formbilayer structure of membranes.

colamine

choline

serine

Hydrophobic “tail”

Hydrophilic “head” Х

69

70

Neutral fats digestion is a step-wise hydrolysis of triacylglycerols (TAG) by pancreatic lipase (lipolysis).

only emulsified fats;active pancreatic lipase (pH 7.5 - 8);colipase (pancreatic protein);bile.

Lipid digestion needs:

Bile acids are synthesized from cholesterol in the liver. They conjugate with glycine or taurineforming bile

salts (conjugated bile acids) and enter the gallbladder as a compound of bile and then to the small intestine.

Bile acids functions:

emulsifica-tion of fats;

lipase activation;

participation in absorption of lypolyticproducts.

Sulphocholeic acid(conjugated bile acid)

3 7

12

Cholic acid

Bile acid

TAG

Hydrophilic side

water

Hydrophobic side

ОН

ОННО

cholic

chenodesoxycholic -Н

desoxycholic -Н

Bile acids Positions of –OH: С-3 С-7 С-12

pancreatic lipase and colipase

pancreas

Gall-bladder

stomach

duodenum

Small intestine

Bile salts

Food fats

micelle

Bile acids

Triacylglycerol (TAG) Diacylglycerol (DAG) β-Monoacilglycol (MAG) Glycerol

71

Gall-bladder

Foodfats

myocyte oradipocyte

Capillary

Small intestine

Smallintestine

ApoСII

stomachрН 1,5-2

Unfavorable conditions for digestion of lipids

1. Bile salts emulsifyfood fats in the small intestine

and form mixed micelles

2. Pancreatic lipaseactivated by colipase and bile salts

cleaves TAG to FFA and β-MAG (hydrolysis to glycerol occurs in small degree)

3. FFA and MAG are absorbedin the small intestine as micellesincluding diphilic bile acids,phospholipids and cholesterol

6.TAG, phospholipids, cholesterol and apoproteinsfrom epithelial cells of the intestinal mucosa are “packed” in chylomicrons ( lipoproteins that transport exogenous fats from the intestine to tissues).

7.Chylomicrons carry dietaryTAG through the lymphatic system and blood vessels to different

tissues such as adipose tissue,skeletal muscles, heartand others.

FFA pass into adipocytes,myocardium,skeletalmuscles, mammaryglands and

other tissues.

10. In the cells FFA are oxidized to the end products with the formation of ATP(in myocytes) or they are used in TAG

synthesis for further accumulation (in adipose tissue).

Stages of Digestion, Absorption and Transport of TAG

chylomicron

8. LP-lipase is activated byapoprotein apoC II. The enzyme cleaves TAG incorporated in chylomicrons to FFA and glycerol. This reaction occurs on the endothelium of adipose tissue capillaries.

Lipoprotein (LP) lipase

11. Some FFA are combined

with albumin for their

transportation in the blood.

4. Micelles decompose in the intestinal mucosa. FA and MFA are resynthesized to specific for human body TAG.

FFA

TAG

5. 90% of bile acids are absorbed from the intestine to blood, then flow to the liver where they are used for biliation once again (entero-hepatic circula-tion has 7-8 cycles per day).

ATP

TAG

FFA

Bile acids

72

Glycerophospholipids are digested by phospholipase which hydrolyze the ester bonds in a molecule of glycerophospholipid.

Abnormal digestion and absorption of fats (in pancreatitis, cholelithiasis, intestinal diseases) result in steatorrhea, that means the excretion of large amounts of undigested fats with the feces.

Digestion of Glycerophospholipids

Products of phospholipids digestion :phospholipase А1

phospholipase А2

phospholipase С

phospholipase D

R1СООН – unsaturated fatty acid

R2СООН – unsaturated fatty acid

phosphatidyl-inositol

diphosphateglycerol

Н

Inositol diphosphate (IF2)

Phosphoric acid

Cholesteryl ester

Cholesteryl esterhydrolase

RСООН

+

Н2О

Pancreatic phospholipase A2 enters the small intestine in the inactive form. It is activated by trypsin by means of partial proteolysis. As a result, it produces toxic lysophospholipids which are not accumulated since the intestinal phospholipases A1, C and D attack phospholipids almost simultaneously.

Digestion of Cholesteryl Esters

Cholesteryl esters received with food of animal origin (plant steroids are not assimilated), are splittedby the intestinal or pancreatic cholesteryl ester hydrolase ase to FFA and cholesterol.

Cholesterol

73

Resynthesis of Specific Fats in the Intestine

MAG entered in the intestinal wall may be converted in two ways:- through lipolysis to glycerol and FFA;- through specific TAG resynthesis involving two molecules of acyl-CoA:

CholesterolPhospho-lipids

TAG and cholesteryl esters

ApoproteinsChylomicrons are the major molecular forms in which resynthesized TAG pass through the lateral membrane of enterocytes. Through the lymphatic vessels chylomicrons enter the lymphatic duct and finally in the blood to transport dietary fats to tissues.Chylomicrons are the largest lipoprotein particles composed of lipids and proteins. They have a diameter of 100-1000 nm. Chylomicrons are formed in the intestinal mucosa. They contain mainly TAG(90%) and the hydrophobic cholesteryl esters located in the core of the particle , as well as a small amounts of phospholipids, free cholesterol and proteins that form the amphipathic membrane.

Transport forms of exogenous lipids are chylomicrons.

The main function of chylomicrons is to transport food TAG from the intestine into the tissues through the bloodstream. The concentration of chylomicrons in the blood plasma usually reaches its maximum 3-6 hours after ingestion of fatty-reach food and then gradually decreases. The rate of particles removal from the plasma is quite high. In healthy people who have been starved for 12 hours chylomicrons are not detected. This is possible due to the action of lipoprotein lipase. This enzyme is located on the endothelial cells of capillaries of different tissues (fat, skeletal muscles, heart). Due to apoprotein apoCIIchylomicrons are recognized by lipoprotein lipase. The enzyme catalyzes the hydrolysis of TAG to glycerol and FFA in chylomicrons. Due to the receptors to apoproteinE chylomicron remnants are taken up by the liver, where they are finally decomposed.

Triacylglycerol (TAG)Diacylglycerol (DAG)β-monoacilglycerol (MAG)

74

FA

ТАG

Inactiveadenylate cyclase

Hormone(epinephrine, glucagon)

Activeadenylate cyclase

АТP

cAMP

Inactiveproteinkinase

Activeproteinkinase

InactiveTAG lipase

АТP

DАG МАG glycerol

FA FA

Active TAG lipase

Intracellular Lipolysis

Adipose tissue(ТАG depot)

Musclesliverheart

ATPСО2 Н2О

TAG inlow-density lipoproteins

ТАG in chylomicrons

Apoproteins

As stated above, TAG are ingested as components of animal and plant food. After digestion, absorption and biochemical transformations in enterocytes, blood and liver TAG are deposited in adipose tissue. Less amounts of TAG are stored in other organs. The accumulation of TAG in adipose tissue is the most effective mechanism of a metabolic energy accumulation. Due to the high con-tent of hydrogen atoms TAG oxidation generates much more energy than that of carbohydrates and proteins.

TAG Catabolism

TAG can perform its energy function only after splitting into free fatty acids and glycerol by intracellular lipolysis. Mobilization of free fatty acids from adipose tissue occurs under the action of lipases (E1, E2, E3). The activity of the 1-st enzyme (E1) is regulated by hormones. It is a hormone-sensitive TAG lipase. Norepinephrine (under stress) and glucagon (during starvation) are activators and insulin is the inhibitor of E1.

Е2 Е3

Hormone-sensitive TAG lipase activity is regulated by a cascade mechanism of hormonal regulation leading to phosphorylation of the enzyme. The liberated FFA are the substrates for the oxidation in many tissues such as heart, skeletal muscle, liver except brain.

Е1

75

carnitine

RCOOH + HSKoA R-CO- SKoAAcyl-CoA synthetase

АМP + PPАТP

P PDihydroxyacetonephosphate

Glycerol Glycerol3-phosphate

АDP NAD+ NADH

Glycerol-3-phosphate

dehydrogenase

Glycerol kinase PGlyceraldehyde-3-phosphate

Acetyl-CoA

Pyruvate

CAC

Н2О СО2

Oxidation of Glycerol

2

Glycerol is activated by phosphorylation (1 ATP molecule is needed). After that glycerol 3-phosphate is dehydrogenated resulting in the formation of dihydroxyacetone phosphate and NADH. The latter transfers hydrogen atoms into the mitochondrial respiratory chain. Oxidation of NADH produces 3 ATP molecules by oxidative phosphorylation. Dihydroxyacetonephosphate enters aerobic glycolysis in which it is converted into glyceraldehyde-3-phosphate that is oxidized in the second stage of aerobic glycolysis to pyruvate. It results in production of 3 ATP molecules by oxida-tive phosphorylation and 2 ATP molecules by the substrate level phospho-rylation. Under aerobic conditions pyruvate enters the common final catabolic pathway: 3 ATP molecules are produced due to the oxidative decarboxylationof pyruvate and 12 ATP molecules are generated in the CAC and the mitochondrial ETC. Thus, the oxidation of glycerol to the end products produces 22 ATP molecules.

ATP

Oxidation of Free Fatty Acids

20

1. Activation of FFA in a cytosol

2. Transfer of acyl-CoA from the cytosol to the matrix of mitochondria by carnitine

Oxidation of FFA occurs in the mito-chondria, but the inner mitochon-drial membrane is impermeable to acyl-CoA. That is why acyl is transported into the mitochondria in complex with carnitine, based on the shuttle principle.

ATP

ATP

HS-CoA acylation coenzyme

FFA +CoA

Acyl-CoAAcyl-CoA

Acyl-carnithine

Acyl-carnithine

Acyl-CoA

76

S-CоА

S-КоА

S-CоА

S-КоА

S-CоА S-CоА

FAD

FADН2

acyl-CоА-

dehydrogenase

Enoyl-CоА-hydratase

ETC

β-hydroxyacyl-CоА-dehydrogenase NАD+

NАDНН+ In CAC

НS-CоАthiolase

palmitoyl- CоА

Acyl- CоА Acyl -CоА

β-ketoacyl-

S-CоАenoyl-

β-hydroxyacyl-S-КоА

β-Oxidation of Fatty Acids

CAC

Stage 1 Stage 2

Stage 3

NАDН, FADH2

Electron transport chain

ADP+P

β-Оxidation8 Acetyl-CоА

Catabolism of fatty acids (FA) proceeds in three stages. At the 1-st stage(β-oxidation) FA is subjected to the oxidative degradation with the removal of a two-carbon atoms fragment as acetyl-CoA, starting from the carboxyl end of the oxidized acyl.

For example, the oxidation of palmiticacid (n=16 carbon atoms) yields 8 (n / 2) molecules of acetyl-CoA which take part in the 2-nd stageof FA oxidation, or in the CAC. At this stage the acetyl groups are oxidized to CO2 .Hydrogen atoms are delivered into the mitochondrial respiratory chain.

At the third stage of FA catabolism the hydrogen atoms from NADH and FADH2 enter the ETC and reduce oxygen to water. Fats are the main source of metabolic water. The energy of FA catabolism is converted to ATP. The oxidation of 8 acetyl-CoA molecules forms 8x12 = 96 ATP molecules. Oxidation includes 7 cycles, each one produces 5 ATP molecules due to the oxidation of NADH and FADH2: 7x5 = 35 ATP molecules. The net result: 141-1 = 140 ATP molecules.(* 1 ATP molecule is needed for FA activation).

the number of oxidation cycles

(n/2-1)=7

2

1

3

4

These four consecutive reactions (β-oxidation cycle) are repeated unless the entire fatty acid containing an even number of carbon atoms (n) will be splitted into(n/2) molecules of acetyl-CoA:

2АТP

3АТP

АТP

76

С12

С10

С8

С6

С4

С14acetyl-CoA

acetyl-CoA

acetyl-CoA

acetyl-CoA

acetyl-CoA

acetyl-CoA

acetyl-CoA

76

77

SCоА

SCоА SCоА

β-hydroxybutyrate

acetoacetate

аcetoacetyl-SCоА

2 аcetyl-SCоА

acetyl-SCоА

НSCоА

Ketone bodies

CACоxaloacetate

hepatocyte

glucose

Lipid drop

Glucose is exported as a fuel for brain and other tissues.

Acetoacetate and β-hydroxybutyrateare exported as a source of energy for skeletal muscles, heart and brain.

CAC

Н2ОСО2

NAD+

NADН

succinyl- SCоА

succinate

НSCоА

β-hydroxybutyratedehydrogenase

thiolase

β-ketoacylCoAtransferase

Metabolism of Ketone Bodies

Ketone BodiesUtilization

Ketone bodies are synthesized exclusively in the liverbut they are never used there. The 1-st reaction coours in a cytosol, the rest ones are in mitochondria of hepato-cytes. Ketone bodies diffuse into blood and are used as alternative fuel in a heart, skeletal muscles, cortical layer of suprarenal glands and others. In starvation ketonebodies are used by the brain.

SCоА SCоА2 аcetyl-SCоА

acetoacetyl-SCоАSCоА

SCоА

аcetyl-SCоА

Β-hydroxymethylglutaryl-SCоА (HMG- SCоА)

acetoacetate

аcetone β-hydroxybutyrate

β-hydroxybutyratedehydrogenase

NADН

NAD+

кетоновые тела

аcetyl-SCоА

thiolaseНSCоА

HMG- SCоА-synthase НSCоА

HMG- SCоА-lyase

Synthesis of Ketone Bodies

In norm concentration of ketone bodies in blood is not high (10-20 mg/l), but it rises radically in fasting and diabetes mellitus. In these states ketonemia and ketonuria are developed.

АТP

кетоновые телаKetone bodies

78

Synthesis of endogenous cholesterol takes place mainly in the liver (50-80%), intestine (10-15%), as well as in skin(up to 5%), in the cortex of suprarenal glands and in the reproductive organs. Biosynthesis of cholesterol starts from acyl-CoA. С27sterin carbon skeleton is formed by binding of С2-units in a long and complex chain of reactions.Cholesterol biosynthesis can be divided into 4 stages:

In the first stage mevalonate (C6) is formed from 3 molecules of acetyl-CoA. The first reactions are totally similar to the ketone bodies synthesis up to HMG-CoA but they take place in hepatic cells cytoplasm. Then HMG-CoA is reduced to mevalonic acid. Regulatory enzyme of this synthesis is HMG-CoA reductase. Its coenzyme is NADPH +.The activity of this enzyme is inhibited by cholesterol and low density lipoproteins (LDL) by o negative feedback regulation. Also HMG-CoA reductase is inhibited by phosphorylation.

At the second stage mevalonate is converted into "active isoprene“, or isopentenyl-diphosphate.

At the third stage six molecules of isoprene are polymerized to form squalene (C30).At the fourth stage squalene is cyclized to lanosterol (C30), from which three methyl

groups are splitted off and cholesterol is formed (C27).

Synthesis of Cholesterol

mevalonic acidβ-hydroxymethylglutaryl-CоА (HМG- CоА)

HМG –CоА reductase

2NADP+

Н2ОН

squalene

lanosterol

глюкозаFFA

2NADPН+

Glucose

PPPcholesterol

NADPН+

NADPН+

biomembrane

Bile acids

Steroid hormones

vitamin D3

isopentenyldiphosphate

thiolase

2 acetyl-SCoA

SCoA

SCoA

SCoA

SCoA

SCoA

Acetoacetyl-SCoA

HMG-CoAsynthase HSCoA

acetyl-SCoA

79

cholesterol

cholesterolester

Phosphatidylcholine (lecithin)

lysophosphatidylcholine

lecithincholesterol-acetyltransferase

(LCAT)

Cholesterol Pool

Intake with food(exogenous cholesterol)

0,3-0,5 g a day

Synthesis from acetyl-CоА(endogenous cholesterol)

1 g a day

Synthesis of steroid hormones ~40 mg a day

synthesis and excretion in bile acids~ 1,0-1,3 g a day

synthesis of vitamin D3

~10 мg a day

excretion in feces~0,5-0,7 g a day

Excretion insebum~ 0,1 g a day

Structure of membranes and lipoproteins

The pool of cholesterol consists of: free cholesterol that is the largestconstituent which includes cholesterol of cell

membranes and cholesterol of blood lipopro-teins membranes.

cholesteryl esters that are hydropho-bic molecules which are the storage form of cholesterol in the body. They are located inthe cytosol of adipocytes and inside lipoproteins. Cholesteryl esters are formed byesterification of cholesterol by unsaturatedfatty acids mainly from lecithin. This reaction needs the enzyme lecithin:cholesterol acyltransferase (LCAT). The another way to produce cholesteryl esters is possible due to the acylationof cholesterol by acyl-CoA:cholesterol acyltransfe-rase (ACAT).In adults a body contains ~ 140 g of choleste-

rol, 90% of it is in the tissues, especially nerve, muscular, connective and adipose ones as well as in the adrenal glands. Another 10% of cholesterol is found in blood plasma and lymph as a compound of lipoproteins (mostly as esters). In ageing cholesterol accumulates in the body.

Cholesterol is a very important substance in the human body. In addition to the structural function as a component of cell membranes it serves as a precursor of many biologically important compounds such as steroid hormones, preventing rickets vitamin D3 and bile acids.

80

Lipoproteins are transport forms of lipids.

triacylglycerols

cholesterol

phospholipids

аpolipoproteins

CHM VLDLP LDLP HDLP

Lipid transport from the intestinal cells

Transport of lipids which are formed in the liver

Transport of cholesterol to the tissue

аtherogenic

Transport of cholesterol from tissues into the liverantiatherogenic

Lipoproteins are separated into several classes by ultracentrifugation and electrophoresis. The density of lipoprotein particles is dependent on the ratio of apolipoproteins to lipids: the more protein content, the higher the density of lipopro-teins. Electrophoretic mobility of lipoproteins depends on the amount and amino acid compositi-on of apolipoproteins.

аpоВ-100phospholipids

ТАGcholesteryl esters

cholesterol

All lipids are circulated in a blood plasma as macromolecular complexes termed lipoproteins. Lipoproteins (LP) are spherical particles consisting of a hydrophobic core and an amphipathic membrane. The core inccludes nonpolartriacylglycerols (TAG) and cholesteryl esters. The lipoproteinmembranes consist of polar lipids such as cholesterol and phospholipids. The charged ends of these molecules are turned outwards. Moreover, the membranes include proteins, or apolipoproteins, noncovalently associated with phospholipids and cholesterol: apoproteins A, B, C, D, E.

Chylomicrons (х60000) VLDL(х180000)

LDL (х180000) HDL (х180000)

All apolipoproteins are not only obligatory structural compounds of lipoproteins. They also activate certain enzymes. For example, apolipoprotein C-II activates extrahepaticlipoprotein lipase, apolipoproteins A-I and C-I activate lecithin:cholesterol acyltransferase(LCAT). Apolipoproteins serve as a “visiting card" of lipoproteins because they are ligandsfor the receptors to lipoproteins.

very low densitylipoprotein

low-densitylipoprotein high-density

lipoproteinchylomicrons

Functions of

Lipoproteins

81

HDL

Reverse cholesterol

transport to the liver

liver

LDL

Extrahepatictissues

intestine

CHM

CHM

remnantsVLDL

remnants

capillary

FFALipoprotein lipase Adipose tissue,muscles

Nascent HDLfrom the liver and intestine

All lipoproteins are involved in the transport of cholesterol and its esters in blood and in their distribution among tissues.

Chylomicrons (CHM) transport exogenous cholesterol from the intestine to the liver through the blood stream.

VLDL are formed in the liver (in small quantities - in the intestine) and transport both exogenous and endogenous cholesterol with other lipids into tissues. These types of lipoproteins (VLDL and CHM) are short-living (half-time is 1,5 - 2 hours).

Blood plasma after intake of fatty food

Blood plasma after starvation

Lipoprotein lipase (LP lipase) located on the surface of the endothelial cells cleaves TAG in chylomicrons and VLDL converting them into chylomicron remnants and VLDL remnants. The latter are named intermediate density lipoproteins (IDL). Chylomicron remnants and some IDL are taken up into the liver for further catabolism.Another portion of IDL serve as the precursors for LDL. VLDL, IDL and LDL have apolipoprotein B-100 that is the ligand for LDL receptors. Low-density lipoproteins are cholesterol-rich particles. This is the main class of lipoproteins that transport cholesterol to the liver and extrahepatic tissues for further use. LDL are considered to be atherogenic, because they are associated with development of atherosclerosis.The defect of LDL receptors results in familial hypercholesterolemia.Nascent HDL are synthesized in a liver. They extract cholesterol from the cell membranes and provide its reverse transport back to the liver. That is why HDLare considered to be antiatherogenic lipoproteins, because they prevent the development of atherosclerosis.

82

Synthesis of Fatty Acids

The Biosynthesis of Lipids (Lipogenesis)Lipogenesis is aimed at accumulation of metabolic fuels as triacylglycols and synthesis of the structural components of biomembranes such as phospholipids and sphingolipids. Biosynthesis of fatty acids is the essential part of this process . Its maximal activity is revealed in adipose tissue, liver and mammary gland during lactation.

In the body palmitic acid (C16) is predominantly synthesized. The direct donor of two-carbon fragments is acetyl-CoA, which is formed during the aerobic oxidation of glucose in the mitochondria. The reactions of fatty acids (FA) synthesis occur in a cytosol. The inner mitochondrial membrane is impermeable for acetyl-CoA.

SCоА SCоАacetylCоА-carboxylase(biotin)

АТPАDP+P

+ biotin

аcetyl-CоА malonyl-CоА

Transfer of acetyl-CoA from mitochondria into cytosol occurs by means of shuttle mechanism. It includes the formation of citrate from acetyl-CoA and oxalo-acetate. Citrate passes through the mitochondrial membrane and carries acetyl group from acetyl-CoA into cytosol.

At the first stage, the carboxylation of acetyl-CoA with the formation of C3 molecule of malonyl-CoA takes place. The key enzyme of FA biosynthesis is acetyl-CoA carboxylase, which needs biotin (vitamin H) for prosthetic group.

At the 1-st stage of this reaction biotin is carboxylated, and ATP is used as the source of energy. At the 2-nd stage carboxyl group is transported from carboxybiotin to acetyl-CoA forming malonyl-CoA.

In mitochondria citrate is the major substrate for the CAC, but it may leave mitochondria for cytosol in case of excess consumption of food carbohydrates.

Oxaloacetate returns to the matrix of mitochondria by special shuttle system, including its reduction to malate, which can pass through the mitochondrial membrane. Another way for oxaloacetate to come back is its gradual conversion to pyruvate that can pass through the mitochondrial membrane. The advantage of the last reaction is production of NADPH+ which is required for fatty acids synthesis.

acetyl-CоА

oxaloacetate citrate citrate

acetyl-CоА

оxaloacetate

pyruvate

malate

pyruvate

СО2

matrix cytosol

citrateinsulin

NADP+

NADPН+

Palmitoyl-CоА

83

A fatty acid synthase (palmitoylsynthase) is a multienzyme complex which is involved in the synthesis of fatty acids. It consists of several enzymes catalyzing the specific reactions of fatty acid synthesis and acyl-carrying protein (ACP). This complex has two SH-groups: 1) the cysteineresidue (Cys-SH) that links the acetyl residue and 2)4'-phosphopantethenic group (Pan-SH) which serves to bind malonyl residue. This part of the enzyme functions as a "long arm that fixes the substrate and passes it from one reaction centre to another one.

The spatial association of several consecutive reactions in a multienzyme complex has a number of important advantages over individual enzymes such as prevention of competitive reaction, coordination of successive reactions. All enzymatic reactions are very effective.

Palmitoyl synthase provides progressive lengthening of the carbon chain by two carbon atoms (C2, C4, C6 and so on) to form 16-carbon acyl residue linked to the SH- group of ACP. Cleavage of the palmitoyl-ACP carried out by hydrolysis. This yields free palmitic acid and palmitoylsynthase.

β-ketoacyl-АCP-synthase

КS МТ

КR

HDЕR

АТ

еnoyl-АTP-reductase

АCP

SН

SН

malonyl-CоА-АTP-transferase

β-hydroxyacyl-АTP-dehydratase

β-ketoacyl-ATP-reductase

Acyl-carryingprotein

acetyl-CоА-АCP-transacetylase

Fatty acid synthase (palmitoyl syntase):

84

CоА

НSCоА

НSCоА

SCоА

NADPН+

NADP+

NADP+

NADPН+

АCP

АCP

АCP

АCPАCP

АCP АCP

АCP

КS

КS

КS

КS

КS

КS

КS

КS

МТ

МТ

МТ

МТМТ

МТ

МТ

МТ

КR

КR

КR

КR КR

КR

КR

КR

HD

HD

HD

HD HD

HDHD

ЕR

ЕR

ЕR

ЕRЕR

ЕR ЕR

АТ

АТ

АТ

АТАТ

АТ АТ

АТHDЕR

1condensation

reduction2 3

4

5

dehydration

reduction

transfer of butyrylfrom the SH-group of ACP onto the SH-group of the enzyme

fatty acid synthasecarrying a methyl groupand malonyl group

аcetyl-CоА

malonyl-CоА

β-ketobutyryl-ATP

Butyryl-ATP

β-hydroxybutyryl-АTP

The cycle is repeated

Acetyl transfer

malonyltransfer

As a result, the synthesis of onepalmitate molecule consumes :

1 molecule of acetyl-CоА,7 molecules of malonyl-CоА14 molecules of NАDPН + Н+;thus formed:

7 molecules of СО2, 6 molecules of H2O, 8 molecules of НSCоА14 molecules of NADP+

The sequence of enzymatic reactions of FFA synthesis:

85

Synthesis of Phosphatidic Acid

85

Glycerol-3-phosphate

glycerol

Phosphatidic acid

SCоАНSCоА

SCоАНSCоА

Glucose

glyc

olys

is

Dihydroxyacetonephosphate

GlucoseAcetyl-CоА

АТP

Triacylglicerols Phospholipids

АDP

glycerolkinaseglycerolphosphatedehydrogenase

NADН+

NAD+Liver

LiverAdipose tissue

Muscles

Fats are the most compact form of energy storage. Therefore a portion of the glucose coming from food is converted into fat. This process is activated by insulin (in the absorptive period). It has the the maximal activyty in the liver and adipose tissue.

Synthesized from acetyl-CoA, fatty acids do not remain in a free state, they are activated into the acyl-CoA with the following use for syn-thesis of TAG and PL.

Phosphatidic acid is a common precursor in the synthesis of triacylglycerols (TAG) and phospholipids (PL).

acylation of glycerol-3-phosphate in the 1-st and 2-ndpositions

86

Phosphatidic acid

3

3diacylglycerol

triacylglycerol

Н2О

P

phosphatidylserine

phosphatidylethanolamine

Phosphatidylcholine (lecithin)

S-аdenosyl-methionine

S-adenosyl-homocysteine

(-СН3)

CDP-choline CMP

CDPCDP-diacylglycerol

CTP

PP

Neutral fats ( TAG) perform mainly energy function. The function of phospholipids (PL) is mainly structural: they are compounds of the cell and lipoprotein membranes.TAG and PL are synthesized in a liver not only for its own needs but also for "export“, or for other tissues, where they come through the blood vessels as a constituents of lipopro-teins. The deficiency of lipotropic factors, or factors preventing the development of fatty liver, such as choline, methionine, serine and others results in impairment of PL synthesis.If synthesis of phospholipids is disturbed it causes the accumulation of TAG in hepatocytesand development of fatty liver.

The drug "Heptral" used to treat liver diseases, is the S-adenosylmethionine.

serine

CМP

В12

CoACoA

87

The Pathology of Lipid Metabolism

Primary hyperlipoproteinemias are hereditary molecular diseases associated with the disturbance of apolipoproteins synthesis. The concentrations of correspondent lipoproteins is increased in the blood of these patients. This group of diseases is rather rare and it has low frequency in human population.

Secondary hyperlipoproteinemias are diseases characterized by high level of lipids. Not only hereditary factors, but also life-style, diet, age, sex and other factors may result in development of secondary hyperlipoproteinemias. They include obesity, atherosclerosis, diabetes, hypothyroidism.

Atherosclerosis is a systemic disease, the main manifestation of which is the deposition of lipids in the intima of blood vessels, or formation of so called lipid plaques. The main components of plaques are cholesterol and its esters. Plaques cause narrowing of blood vessels, and therefore an insufficient blood supply to organs and tissues, which may lead to the development of coronary heart disease, myocardial infarction or stroke. The molecular basis of atherosclerosis is a high level of cholesterol, but crucial is the ratio LDL / HDL. The development of atherosclerosis is caused by the increased amount of both atherogenicLDL and chemically modified LDL . Drug therapy of atherosclerosis includes the following medicine:

statins: they are competitive inhibitors of HMG-CoA reductase decreasing synthesis of endogenous cholesterol (pravastatin, lovastatin);

drugs binding with bile acids and increasing the excretion of bile acids from the body (colestipol, cholestyramine);

fibrates: they activatе lipoprotein lipase and reduce the formation of VLDL (fenofibrate);antioxidants: they inhibit the peroxide modification of lipoproteins (vitamin E, probucol);nicotinic acid: it reduces the formation of VLDL and increases the level of

antiatherogenic HDL.

Obesity means the increased deposition of neutral fats in adipose tissue leading to the increase of body weight.

Primary obesity is caused by the excess amount of food rich in calories in diet. Genetic factors play a certain role in the development of obesity. There is an obesity gene known as ob-gene, encoding protein leptin. Leptin functions to decrease appetite and food consumption as well as to stimulate lipolysis. Mutations in ob-gene lead to the obesity.

Healthy mouse (from the right) and mouse with obesity (from the left)

Secondary obesity is caused by an associate illness, mostly of hormonal nature.

88

Multiple Choice Questions

Test 1The elevated level of ketone bodies has been revealed in blood of a patient with diabetes mellitusand metabolic acidosis. What substance is the precursor of ketone bodies synthesis?

A. Methylmalonyl-CoAB. Succinyl-CoAC. Propionyl-CoAD. Malonyl-CoAE. Acetyl-CoA

Test 2To increase physical strength and stimulate energy metabolism carnitine was recommended to a sportsmen. What biochemical process needs this substance?

A. Transport of fatty acids into mitochondriaB. Ketone bodies synthesisC. Fatty acids synthesisD. Tissue respirationE. Formation of lipoproteins

Test 3Mobilization of triacylglycerols from adipose tissue is a biochemical process yielding glyceroland free fatty acids. Free fatty acids are transported in the bloodstream by ...

A. Low density lipoproteinsB. GlobulinsС High density lipoproteinsD. Serum albumins E. Chylomicrons

Test 4Laboratory investigation of the patients’ blood plasma performed 4 hours after a consumption of fat-rich diet revealed that plasma was very turbid. The most credible cause of thisphenomenon is the increased amount of ..... in the plasma.

A. High density lipoproteinsB. ChylomicronsC. Low density lipoproteinsD. Intermediate density lipoproteinsE. Chylomicron remnants

Test 5A 69-year-old man is prescribed a drug that inhibits the activity of the enzyme HMG-CoA reductase. Which metabolic pathway needs this enzyme?

A.Beta-oxidation of fatty acidsB. Cholesterol synthesisC.Triacylglycerols synthesisD.Fatty acids synthesisE. Phosphadidic acid synthesis

KEYS: 1 – E; 2 – A; 3 D; 4 – B; 5 – B.

89

METABOLISM OF PROTEINS

peptide bond

Proteins arebiological macromolecules composed of amino acidresidues united by a peptide bond.

The structure of proteins is divided into 4 levels of organization:

Primarystructure

is the order in which amino acids are bound together in polypeptide chain.Chemical bonds:

peptide bonds.

Secondary structure is spatial arrangement ofpolypeptide chain as alpha-helix or beta-sheet.Chemical bonds:hydrogen bonds.

Tertiary structureis three dimensional structure

of a functional protein.Chemical bonds:hydrogen, disulfide, electro-static, hydrophobic bonds.

Quaternary structure is association of a few subunits

(protomers) intooligomers.

Chemical bonds:hydrogen, electrostatic, hydrophobic bonds.

Protein metabolism plays a very important role in all living organisms because proteins perform essential biological functions such as structural, catalytic, hormonal and defensive.They serve as carriers of vitamins, oxygen, carbon dioxide. Protein metabolism is associated with metabolism of amino acids. Twenty kinds of amino acids are found in human proteins. Each one of them has its own functions and metabolism.

.

Н2N-CH-C N-CH-C N-CH-C N-CH-C N-CH-C N-CH-C N-CH-C N-CH-C-OH

O O O O O O O OH H HHHHH

R R RR R R RRR

90

Protein starvation (Kwashiorkor)

Anaemia

Edemas

Fat liver

Abnormality of protein digestion

Low protein intake

Growth delay

Decreased hemoglobin synthesis

Decreased albumin synthesis

Pancreatic cells atrophy

Decreased lipoprotein synthesis

снижен синтезклеточныхбелков

снижен синтезклеточныхбелков

снижен синтезклеточныхбелков

снижен синтезклеточныхбелков

Decrease of intra-cellular proteins synthesis

Twenty kinds of amino acids are used for synthesis of any proteins. According to the nutritional requirements there are two groups of amino acids: essential (They cannot be synthesized in the body. Diet is the only source of the essential amino acids.) and non-essential amino acids (They are synthesized in the body.). The nutritional importance of proteins is based on the content of the essential amino acids.

Proteins are very important nitrogen-containing molecules. The term “nitrogen balance” is used to assess the difference between total nitrogen intake (nitrogen in; I) and total nitrogen loss (nitrogen out; U) in urine, feces and perspiration.

positive nitrogen balance (I > U): it is observed in growing children, duringrecovery after serious illness, in pregnant women;

negative nitrogen balance (I < U): it is observed in states with acceleratedprotein catabolism, ageing, cancer, after surgery, in starvation, in kwashiorkor or marasmus, incritical illnesses;

nitrogen balance (I = U): it is observed in adult healthy people with balanced diet.

Approximately 400 g of human proteins are cleaved to amino acids daily, and the same quantity is produced. Of the liberated amino acids, 25 % are degraded irreversibly, that is why this portion of total amino acids should be compensated by food proteins. To maintain health, adults require 0,7 g of proteins per 1 kg of body weight (100-150 g per day). Deficiency of even small quantity of proteins in food results in grave consequences.

91

Protein digestion means hydrolysis (proteolysis) of peptide bonds in a protein molecule resulting in release of free amino acids.

Protein digestion is carried out by means of hydrolases that are called proteolytic enzymes(proteases or peptidases). They are produced as the inactive enzymes (zymogens orproenzymes). These enzymes are activated by partial proteolysis to cleave N-terminalpeptide and to form the active centre of the enzymes.

The active enzyme can rapidly activate its own zymogen to make the zymogen molecules active (autocatalysis).

PROTEIN DIGESTION

…

peptide bondС-end of chain

N-end of chain

amino acids residues

Inactive enzyme(proenzyme or zymogen)

Active enzymeN-terminal peptide

autocatalysis

Н2О

Proenzymes synthesis ( in stomach, in pancreas) and their activation (in stomach cavity, in small intestine cavity) do not occur in the same place. Such spatial separation prevents stomach and pancreas from autodigestion.

Untimely activation of proenzymes in stomach and in pancreas takes place in gastric ulcer (activation of pepsin) and in acute pancreatitis (activation of trypsin).

92

Proteolytic enzymes can be used as drugs for medical treatment of abnormal secretion of gastric juice, acute pancreatitis and other diseases of gastrointestinal tract. Proteases are also very important for treatment of festering wounds because they cleave proteins of purulent contents.

All peptidases are enzymes with relative substrate specificity: they cleave all proteins, but each enzyme hydrolyzes peptide bonds only between certain amino acids.

Pepsin cleaves peptide bonds formed by aromatic amino acids (phenylalanine, tyrosine) or between leucine and glutamate.

Trypsin cleaves the peptide bonds, the carbonyl group of which is contributed by arginineor lysine.

Chymotrypsin is specific for peptide bonds containing phenylalanine, tyrosine, tryptophan.Elastase attacks bonds next to small amino acid residues (glycine, serine, alanine).

Dipeptidases

Carboxy-peptidase

AminopeptidaseEndopeptidases:pepsintrypsinchymotrypsinelastase

amino acid residue

endopeptidasesexopeptidases

Proteolytic enzymes are subdivided into two groups according to the site of their action.Enzymes which act on the peptide bonds of terminal amino acids are exopeptidases.They are subdivided into aminopeptidases (act on N-terminal amino acid),

carboxypeptidases (act on C-terminal amino acid), di- and tripeptidases that cleave di- and tripeptides.

Enzymes which attack the internal peptide bonds and liberate peptide fragments are endopeptidases. Pepsin, trypsin, chymotripsin, elastase are endopeptidases.

Depending on serine, cysteine or other amino acids presence in their active centre there are serine, cysteine and other proteases.

Premature activation of pancreatic zymogens is prevented by trypsin inhibitor that forms a very stable complex with the enzyme. The internal surfaces of stomach and small intestine are covered by mucins – glycoproteins of mucous membrane that protect the epithelium of gastrointestinal tract from destruction by active enzymes.

93

DIGESTION OF PROTEINS IN STOMACH

denaturation of proteinsmaintenance of рН 1,5 – 2 in gastric juiceactivation of pepsinogenkilling of certain microorganisms

Gastricsin is another proteolytic enzyme of stomach. Its optimal pH is 3, so this enzyme hydrolyzes proteins in decreased acidity of gastric juice.

Hyperchlorhydria – the increased НСl production. Hypochlorhydria - the decreased НСl production.Achylia – absence of both НСl and pepsin in gastric juice

(total achylia or gastrectomy leads to pernicious anemia because of lack of intrinsic factor (Castle factor) – glycoprotein which is used for absorption of vitamin В12).

Abnormalities of Protein Digestion in Stomach:

гипоацидные состояниярН 3 - 5

The main proteolytic enzyme of stomach is pepsin, which is produced in the chief cells of mucous membrane as the inactive proenzyme pepsinogen. Pepsinogen is activated by hydrochloric acid (slow activation) and afterwards by autocatalysis (rapid activation). Mechanism of activation is partial proteolysis. Pepsine is an endopeptidase with relativesubstrate specificity. The enzyme cleaves peptide bonds formed by aromatic amino acids or dicarboxylic amino acids.

Functions of hydrochloric acid:

Lactic acid (lactate) is absent in normal gastric juice. It is produced in malignant tumoursof stomach as the result of anaerobic glycolysis activation.

autocatalysis

Pepsin

НClPepsinogen

Optimal рН for pepsin is 1,5-2

In the stomach of an infant rennin catalyzes the curdling of milk.

This enzyme converts soluble protein caseinogen into insoluble casein which is digested by pepsin.

Hypoacidic statesрН 3 - 5

Н2N-CH-C N-CH-C N-CH-C N-CH-C N-CH-C N-CH-C N-CH-C N-CH-C-OH

O O O O O O O OH H HHHHH

R R Tyrosine RRPhenyl-alanine R R R

94

DIGESTION OF PROTEINS IN THE INTESTINE

The enzymes of pancreatic juice (trypsin, chymotrypsin, elastase, carboxypeptidase) aresecreted into intestine as proenzynes. Their conversion with the following participation in digestion of proteins occur in duodenum for trypsin and in small intesnine for other enzymes. Enterokinase activates conversion of trypsinogen to trypsin. Trypsin catalyzes the formation of chymotrypsin, elastase and carboxypeptidase from their inactive precursors.Aminopeptidase and dipeptidase of the intestinal juice are produced in active state. The pH of intestinal juice is 7,0 – 8,0.The action of all gastrointestinal tract proteolytic enzymes results in full hydrolysis of food proteins and liberation of free amino acids that are absorbed by intestinal epithelial cells. The transport of amino acids occurs against the concentration gradient (active transport). As opposed to proteins amino acids are not antigens and they are not species-specific substances.

PATHWAYS OF AMINO ACIDS CONVERSION

Carbon skeletons of amino acids (alpha-keto acids) may be produced after the removal of their alpha-amino groups by deamination or transamination. Carbon skeletons are used for:

glucose synthesis (gluconeogenesis) - glucogenic amino acids;lipid and ketone bodies synthesis (ketogenesis) – ketogenic amino acids;oxidation to end products (water, carbon dioxide) with energy release. The released amino group as ammonia (toxic substance) is converted to nontoxic urea

(end product of nitrogen-containing substances) and excreted in kidneys.The removal of amino acids carboxy groups as carbon dioxide (decarboxylation) leads to

formation of biologically active substances (biogenic amines) from certain amino acids.

Trypsinogen

Chymotrypsinogen

Proelastase

Procarboxypeptidase

enteropeptidase

Н2N-CH-C N-CH-C N-CH-C N-CH-C N-CH-C N-CH-C N-CH-C N-CH-C-OH

O O O O O O O OH H HHHHH

R RArginineLysine

PhenylalanineTyrosine

Tryptophan RGlycineAlanine R R

Amino-peptidase

ChymotrypsinTrypsin Elastase Carboxypeptidase

95

There are no special storage molecules for proteins like glycogen for glucose or triacylglycerols for fatty acids.Free amino acids represent the amino acid pool of the body. Its sources are:

amino acids released from dietary protein; amino acids released due to degradation of body protein;amino acids synthesized de novo (non-essential amino acids).

AMINO ACID POOL

СО2 , Н2О

АТP

Excretion into feces10g/day

Absorption150 g/day

Amino acid pool100 g

Diet50-100g/day

Protein secretion

50 g/day/

Alpha-keto acids

Ketonebodies

Glucose

Lipids

Digestion

intestine

kidneys

Excretion into urine

PyruvatePhosphoenolpyruvate3-PhosphoglycerateAlpha-keto acidsGlucose

translation

maturation

СО2

Amino acids

Biogenicamines

First of all free amino acids are used for protein synthesis: structural proteins, enzymes, hormones, receptors and clotting factors. Some amino acids serve as precursors for synthesis of biologically important compounds (histamine, serotonin, melanin).

Body protein in adult10000 g

UreaAmmonium salts

Synthesisde novo

Degradation50-100g/day

NН3

modification

96

DEAMINATION OF AMINO ACIDS

Glutamate

α-ketoglutarate

glutamate

dehydrogenase

NADН Н+

NAD+

NН3

The removal of alpha-amino group from the amino acids as ammonia is termed deamination.

All amino acids except lysineparticipate in deamination.

Types of deamination:direct oxidative deamination for

glutamate;direct non-oxidative deamination: hydrolytic for cysteine,intramolecular rearrangementfor hystidine,reductive for serine and threonine;indirect for most amino acids.

Oxidative deamination is the main kind of direct deamination. Glutamate is the amino acid that undergoes direct oxidative deamination.

The reaction is catalyzed by glutamate dehydrogenase (coenzymes NAD+ or NADP+). It takes place in mitochondria of most tissues with maximal activity in liver. This reaction converts glutamate into alpha-ketoglutarate with liberation of free ammonia (toxic substance) with the following detoxication of the latter. Most of amino acids undergo indirect deamination. The first stage of indirect deamination is transamination.

TRANSAMINATION OF AMINO ACIDS

The transfer of an amino group from the amino acid (donor) to alpha-keto acid (acceptor) leading to formation of both new amino acid and new alpha-keto acid without liberation of free ammonia is called transamination.

1-st amino acid 1-st ά-ketoacid 2-nd ά- keto acid

2-nd amino acid

Directoxidativedeamination

97

The main amino groupsdonors in transaminationare glutamate, aspartateand alanine.The main acceptor of

amino groups from different amino acids isα-ketoglutarate.

Transamination of amino acids is catalyzed by aminotransferases (transaminases). These enzymes require coenzyme pyridoxal phosphate (PLP) – a derivative of vitamin В6. During the reaction PLP is linked with amino group of amino acid forming pyridoxamine phosphate. Transamination is reversible. Serum aminotransferases are important for diagnostic and prognostic purposes. Alanine aminotransferase (ALT) activity is increased in acute hepatitis of viral or toxic origin, jaundice and cirrosis of liver. Aspartate aminotransferase (AST)activity is increased in myocardial infarction.These enzymes are considered to be markers forliver and heart, respectively. That is why any damage of liver or heart cell membranes results in increased appearance of ALT or AST in blood serum and elevation of their activity.

Biological importance of transaminationaccumulation of amino groups from different amino acids as glutamate – the only amino acid that undergoes direct oxidativedeamination to release free ammonia for urea synthesis;production of non-essential amino acids;

redistribution of the amine nitrogen in tissues;the initial stage of amino acids catabolism, the first stage of indirect

deamination; possibility for alpha-keto acids to enter different metabolic pathways

(carbohydrate, lipid metabolism).

INDIRECT DEAMINATION

1 – transamination between amino acid and alpha-ketoglutarate with the following formation of glutamate,2 – direct oxidative deamination of glutamate with the following liberation of free ammonia.Glutamate dehydrogenase, catalyzing the 2-nd stage of transdeamination, is controlled by allosteric regulation. Its activity is inhibited by NADH, ATP, GTP. GDP and ADP are activators of this enzyme.

α-Ketoglutarate Glutamate

Amino acid α-Keto acid

NН3

glutamate dehydrogenase

Some amino acids cannot participate in direct deamination, so they undergo indirect deamination, or transdeamina-tion.

α-Ketoglutarate

1

2

98

Аmmonia is a toxic substance. In high concentration it is very harmful to nervous cells. The main causes of ammonia toxicity are the following:

METABOLISM OF AMMONIA

NН3

Amino acids

Nucleotides

Biogenic amines

Putrefaction of proteins

Sources of Ammonia:direct and indirect deamination of amino acids ;inactivation of biogenic amines;deamination of purine and pyrimidine nucleotides;putrefaction of proteins in large intestine.

NН3 + α-Ketoglutarate Glutamate

NADPНН+NАDP+

It leads to elimination of α-ketoglutarate from the pool of amino acids with the following inhibition of metabolic pathways that need participation of α-ketoglutarate. They are:

citric acid cycle (impairment of ATP formation);metabolism of amino acids (transamination impairment). Аmmonia increases synthesis of glutamine in brain. Accumulation of glutamine in the

brain results in elevation of osmotic pressure in nervous cells and brain edema.

Аmmonia binds α-ketoglutarate (reductive amination)

NН3 + Glutamate GlutamineАTP АDP + P

In blood ammonia is represnted as ammonium cation NН4+ :

Accumulation of ammonium cations impairs transport of ions (Na+, К+ ) through cell membranes and failure transmission of nerve impulses.

NН3 + Н+ NН4+,

99

Pathways of Ammonia Disposal

Urea and salts of ammonium are end products of ammonia detoxication. They are produced in liver and kidneys, respectively, and excreted from the body into urine. But ammonia is produced in all tissues, and its production is dramatically increased during long-term exercise load and nervous excitation. To prevent the toxical action of ammonia special transport molecules are created for its delivery to liver and kidneys.

The main transport molecule for ammonia is glutamine. Glutamine is a neutral substance, so it is able to penetrate cell membranes inside the cells.

АDP + PNН3 + Glutamate Glutamine

АTP

glutamine synthetase

In kidneys and liver glutamine is cleaved to glutamate and free ammonia by the enzyme that is named glutaminase.

1. Formation of Transport Forms for Ammonia

NН3Glutamine Glutamate +glutaminaseН2О

Due to this reaction the concentration of ammonia in blood is very low (25-40 mcmoles/l).

As alanine ammonia is removed from intestine and skeletal muscle (glucose - alanine cycle).Activity of glycolytic enzymes in skeletal muscle is

high. But during labour a muscle receives portion of energy from catabolism of amino acids. By transamination amino groups from different amino acids are incorporated into glutamate. Transaminationof glutamate and pyruvate that is produced in glycolysisresults in alanine production, so alanine molecules have amino groups from amino acids and carbon skeletons from glucose.

GlutamatePyruvate

α-Ketoglutarate

Alanine

+ +

Alanine is transported into blood to liver where it underdoes indirect deamination to release free ammonia. After that ammonia is converted to urea for its excretion. Pyruvate is used in gluconeogenesis to

synthesize glucose as the fuel for skeletal muscle.

(α-KG)

urea

excretion into urine

kidneys

proteines

alanine

pyruvate

alanine

glutamate

glucose

glucose

pyruvate glutamate

amino acid

α-KG

α-KGalanine

pyruvate

100

COOH

COOH

CH2

CHH2NNH2

O

NH2C

COOH

CH NH2

(CH3)3

NH

C

NH

COOH

COOH

CH2

CHNH

NH2

COOH

CH NH2

(CH3)3

NH

C

NH

CO2

2АТP

2АDP + Pi

C

O

OH 2 N ∼ P

O H

O

O H

Pi

carbamoylphosphatesynthatase I

Carbamoyl phosphate

ornithinecarbamoyltranspherase

5

1

2

3

4

arginino-succinate

synthetase

arginino-succinate

lyase

arginase

Argininosuccinate

mitochondrialmatrix

cytosol

Arginine

Aspartate

Ornithine

Ornithine

Citrulline

Citrulline

NH2

COOH

CH NH2

(CH3)3

NH2

COOH

CH NH2

(CH3)3

NH

C O

COOH

CH NH2

(CH3)3

NH

C

NH

OH

FumarateCOOH

CH

COOH

CH

Urea

Glutamine

Glutamateglutaminase

Aspartate

Oxaloacetate

ά-KG

ATP

AMP + PPi

From extrahepatic tissues Glutamate

ASTglutamate

dehydrogenase

NH3

HOH

101

2. Biosynthesis of Urea (Ornithine Cycle, Urea Cycle)

Biosynthesis of urea (Krebs – Henseleit cycle) is the main mechanism for the disposal ofammonia from the body in mammals including man.

NН4+

N Н2

Н2N

Н2N-

НN

NН2+

NН3+

N Н

Н2 N C

C

C

C

C

CCО2

ArgininosuccinateFumarate

Aspartate

Ornithine

Ornithine Citrulline

Citrulline

Carbamoyl phosphate

Glutamine

NН2

NН2

C =О

Urea

2АТP

2АDP+P

carbamoylphosphatesynthetase I

АТP

PP

mitochondrialmatrix

cytosol

Biosynthesis of urea occurs only inliver (in mitochondria and cytosol) by conversion of toxic ammonia to non-toxic urea. Urea is a water-soluble and easily excreted compound. Urea accounts for 80 – 90% of the nitrogen containing substances excreted into urine. Urea cycle starts with the condensation of NН4

+ with CО2 to form carbamoyl phosphate.

АМP

ornithinecarbamoyltransferase

arginino-succinatesynthetase

arginino-succinatesynthetase

Arginino-succinatelyase

arginase

Arginine

phosphate

ribose

adenine

АМP

intermediate

102

The molecule of urea has two nitrogen atoms: thefirst nitrogen atom enters the ornithine cycle as ammonia; thesecond nitrogen atom is incorporated into urea from aspartate.

N

N

mitochondrial matrix

cytosol

ornithine

arginine

citrulline ornithine

argininosuccinate

citrulline

fumarate

malate

aspartate

aspartate

fumarate

malateCAC

Urea cycleCycle of aspartateregeneration from fumarate

oxaloacetate

NAD+

NADH

glutamate

NН2

NН2

C =О

urea

Aspartate that is used for urea synthesis, is produced in liver from carbon skeleton of oxaloacetate and amino group of alanine.Fumarate that is produced in urea cycle, is converted to oxaloacetate by means of two reactions of citric acid cycle (CAC), thereby the regeneration of aspartate from fumaratetakes place.

NН3

alanine

α-ketoglutaratecarbamoyl phosphate

pyruvate

-NН2

Synthesis of urea reguires 3 molecules of ATP or energy of 4 high energy bonds: carbamoyl phosphate synthesis needs 2 ATP molecules and energy of 2 high energy bonds; argininosuccinate synthesis needs 1 ATP molecule but energy of 2 high energy bonds (АТP → АМP + P + P).

The liver diseases (hepatitis, cirrhosis) and deficiency of any enzyme of the urea cycle result in hyperammoniemias: elevation of ammonia concentration in blood. Clinical symptoms of hyperammoniemias include vomiting, irritability, avoidance of high-protein foods, intermittent ataxia, lethargy and mental retardation.

3. Synthesis of Ammonium Salts in KidneysAmmonia is transported to kidneys by glutamine. Glutamine is cleaved by glutaminase. Acidosis leads to the activation of glutaminase with the following release of ammonia. Ammonia can be used for the neutralization of acid substances and formation of the ammonium salts (0,5 g /day):

Н+А + NН4+АNН3(acid)

This reaction prevents loss of К+ and Nа+

through urine.

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Glucogenic amino acids can be converted to pyruvate and the intermediates of citric acid cycle (CAC) for glucose synthesis by gluconeogenesis.

Glucogenic and ketogenic amino acidsare used for both glucoseand ketone bodiessynthesis.

Ketogenic amino acids are converted to acetoacetate or acetyl-CoA – precursors for ketone bodies synthesis.

amino acids

CAC

Cycle of aspartateregeneration from fumarate

Urea cycle

carbonskeletons

α-keto acids

urea oxaloacetate

glucose

СО2Н2ОАТP

NН3

carbamoyl phosphate

Syntheses of amino acids,nucleotides

GLUCOSE Ketonebodies

glutamate,glutamine,hystidine, proline,

arginine

Pyruvate

aspartatephenyl-alanine,

tyrosine

valine,methionine,threonineisoleucine

CACtryptophan,

leucine

isoleucine

leucine,lysine

phenylalanine,tyrosine

asparagine,aspartate

α-keto-glutarate

acetoacetateAcetyl-CoA

succinate

fumarate

oxaloacetate

alanine, glycine,cysteine, serine,threonine

Carbon skeletons that are produced from amino acids deamination, can enter the citric acid cycle (CAC) through α-ketoacids for their further oxidation to carbon dioxide and water. But it is not the efficient usage of valuable organic molecules. That is why they are considered to serve mainly for gluconeogenesis.The ammonia produced participates in biosynthesis of nitrogen containing substances or is excreted as urea.

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DECARBOXYLATION OF AMINO ACIDS

СО2decarboxylase

PLP+

Serotonin

Tryptophan

Biogenic amines arephysiologically active

substances

Tryptamine

СО2

Glutamateγ-Aminobutyric acid

(GABA)

СО2

Н2N СН2 RAmino acidН2N СН R

СООН

Epinephrine

Dopamine

TyrosineСО2

DOPA

Norepinephrine

HistamineHistidine

СО2

Hydroxytryptophan

СО2

Н2N СН2 R

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Certain amino acids undergo decarboxylation that means the removal of their α-carboxyl group resulting in liberation of СО2 and formation of biogenic amines – physiologically active substances such as hormones, neurotransmitters and others. Some biological amines are incorporated into other biological compounds. For example, phosphatidylethanolamine (cephalin) contains ethanolamine, coenzyme A contains β-alanine.Decarboxylation of amino acids is catalyzed by α-decarboxylases of L-amino acids. The enzymes are stereospecific, so they act only on L- and do not act on D-amino acids. Pyridoxal phosphate (active vitamin B6 ) is the coenzyme of these enzymes.Over 40 biogenic amines are known, and the most important are the following ones:

Serotonin

γ-aminobutyricacid (GABA)

is a neurotransmitter (stimulator). In mammals the largest amount of serotonin is synthesized in the intestinal cells. Platelets contain serotonin high concentration , but they do not synthesize it. Serotonin is a vasoconstrictor. Serotonin controls the behavioural patterns, sleep, blood pressure, body temperature. It causes the release of gastrointestinal hormones. It is also necessary for the gastrointestinal tract motility.

Dopamineis a neurotransmitter in the brain. The decrease of dopamine production in substantia nigra, locus coeruleus results in Parkinson’s disease. Dopamine is the precursor for the synthesis of norepinephrine and epinephrine.

Norepinephrineis the inhibitory neurotransmitter of the sympathetic nervous system in different areas of brain, but it can excite the brain activity in hypothalamus.Decrease of dopamine and norepinephrine content in nervous cells results in depression.

Histamine

is produced in mastocytes of the connective tissue. Histamine secretion in the bloodstream is stimulated by damage of tissues (trauma, burn). Histamine plays an important role in inflammation. Elevation of histamine concentration leads to edema, vasodilatation and reddening of the skin.Histamine stimulates the secretion of HCl by gastric mucosa and saliva by salivary glands,

promotes pepsin synthesis. Histamine decreases arterial blood pressure, but increases intracranial pressure.Histamine stimulates contraction of smooth muscles in lungs.Excessive production of histamine causes asthma and allergic reactions.In nervous tissue histamine is a neurotransmitter.

is synthesized in the grey matter of the central nervous tissue. GABA is an inhibitory neurotransmitter.GABA opens chloride channels and increases the permeability of post-synaptic membranes. Decreased GABA quantity leads to the neuronal hyperexcitability causing convulsions. The structural analogs of GABA are used in the treatment of epilepsy, disturbance of brain circulation and depression.

Epinephrineis the hormone of the intensive physical labour and stress. Epinephrine regulates carbohydrate and lipid metabolism, causes the increase of blood pressure and heart contraction.

Inactivation of Biogenic Amines

R СН OR СН2 NН2

NН3

monoamine oxidase(FAD)

1. Oxidation and deamination by monoamine oxidase (MAO):

2. Methylation by S-adenosylmethionine: for histamine, epinephrine and norepinephrine:

epinephrine methylepinephrine

Administration of drugs that are МАО inhibitors is used in the treatment of Parkinson’s disease.

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Metabolism of Aromatic Amino Acids in Various Tissues

Phenylalanine

Tyrosine

Homogentisate

Fumarylacetoacetate

Fumarate Acetoacetate

p-Hydroxyphenylpyruvate

phenylalaninehydroxylase

Phenylketonuria

tyrosinetransaminase

Tyrosinemia II

p-hydroxy-phenylpyruvate

oxidase

Tyrosinemia III

homogentisatedioxygenase

Alkaptonuria

fumarylaceto-acetase

Tyrosinemia I

Thyroid gland:tri-, tetraiodothyronines

Adrenal medulla:epinephrine

norepinephrine

DOPA

Skin:melanins

Albinism

NADPH+

NADP+

О2

Н2О

О2

СО2

О2

Dopamine

Phenylalanine is an essential amino acid, tyrosine is considered to be a relatively non-essential amino acid because it is produced only from phenylalanine. Catabolism of these amino acids occurs in liver. Its products, fumarate and acetoacetate, are used both in gluconeogenesis and in ketone bodies synthesis, respectively. So, phenylalanine and tyrosine are glucogenic and ketogenic amino acids.

The defects of the aromatic amino acids catabolism result in different metabolic disorders.

Phenylketonuria is characterized by elevated level of phenylalanine and its metabolites such as phenylpyruvate, phenyllactate, phenylacetate in blood and urea. The main symptoms: mental retardation, tremor, hyperactivity, microcephaly, growth, walking and speech disturbances. Alkaptonuria is characterized by accumulation of homogentisate. Homogentisate forms polymers, that cause urine to darken when exposed to air (on standing). The main symptoms: dark urine (homogentisic aciduria), joint arthritis and black (ochronotic) pigmentation of cartilages.Albinism is characterized by partial or full absence of melanin production. The affected people have white skin, hair, and iris colour. They may have vision defects and photophobia. Sunlight is painful to their eyes. They sunburn easily and do not tan.Tyrosinemia includes several diseases associated with tyrosine accumulation in blood and urine caused by the lack of its

catabolism enzymes activity. The neurological symptoms are the same as for phenylketonuria and tyrosinemia.

107

Metabolism of Glycine and SerineGlucose

3-Phosphoglecerate

Pyruvate

Serine

glutathione synthesis

heme synthesis

purine nucleo-tides synthesis

formation of conjugated bile acids

creatine synthesis

THF N5,N10(СН2)ТHF

СН2ОН serine hydrpxymethyl transferase

Glycine

glycine oxidase

Glycine Glyoxylate

NAD+ NADH+

Oxalate

НСООН

ТHF

N5,N10(СН2)ТHF

MethionineАТP

S-Adenosyl-methionine(SAM)

P+PP

Homocysteineadenosine

S-Adenosylhomocysteine

R

R-СН3

Adenosine Н2О

methionine S-adenosyltransferase

Н3С

СН3

N5(СН3)ТHF

ТHF

Vitamin В12

1

23

4

Metabolism of Methionine

Methionine is an essential amino acid. It serves as the precursor for cysteine synthesis. As SAM methionineis used in transmethylation reactions. Methyl groups are necessary for synthesis of creatine, choline, lecithin, purine and pyrimidine nucleotides, epinephrine. Due to taking part in choline synthesis Met is considered to be the lipotropic substance, because it prevents fat dystrophy of liver.Methyl groups are necessary for xenobiotics detoxification in the liver. Methionine-tRNA participates in the formation of the initiation complex binding to the initiation codon AUG at the beginning of translation (protein synthesis).

methyl-transferases

Glycine and serine are non-essential amino acids, they can be synthesized from glucose. Both amino acids are the precursors for synthesis of different biologically important substances. Glycine and serine are the main sources of one-carbon fragments which are transfered by tetrahydrofolate (THF). One-carbon fragments are used for the synthesis of purine and pyrimidine nucleotides (thymidylate), formylmethionine tRNA (required for initiation of protein synthesis) and other compounds.

108

Multiple Choice QuestionsTest 1

Patient with encephalopathy was admitted to neurological department of the hospital. Correlation of the increasing encephalopathy and concentration of toxic substances absorbed from the large intestine into blood has been revealed. Which substance causes endotoxemia?

A. BiotinB. ButyrateC. AcetacetateD. IndoleE. Ornithine

Test 2Examination of patient with cancer of urinary bladder had revealed high concentration of serotonin and hydroxyanthranilic acid in blood serum. It is caused by excess of ... quantity in the patient.

A. AlanineB. TryptophanC. HistidineD. MethionineE. Tyrosine

Test 3A 13-year-old boy complains about general weakness, dizziness and tiredness. He is mentally retarded. Increased level of valine, isoleucine, leucine has been determined in blood and urine of the patient. Urine of the boy has specific smell. What is the diagnosis?

A. Maple syrup urine diseaseB. PhenylketonuriaC. TyrosinosisD. HistidinemiaE. Alkaptonuria

Test 4Nappies of a newborn have dark spots that are associated with formation of brownish-black pigment from homogentisic acid. Which amino acid has disordered metabolism?

A. TryptophanB. GalactoseC. MethionineD. CholesterineE. Tyrosine

Test 5A patient complains about dizziness, memory impairment and periodical convulsions. It was revealed that these symptoms were caused by the deficiency of the product of glutamic acid decarboxylation. Name this product.

A. SerotoninB. Pyridoxal phosphateC. GABAD. HistamineE. NAD

109

Test 6Subcutaneous injection of the antigen had caused in patient the following symptoms: reddening of skin, edema and pain. The increased level of histamine had been determined in blood of the patient. This biogenic amine is produced from histidine by ...

A. PhosphorylationB. MethylationC. DecarboxylationD. IsomerizationE. Deaminization

Test 7A 2-year-old child with mental and physical retardation, decreased pigmentation of skin and hair, eczema had “mousy” odor. Concentration of catecholamines was low in his blood. After chemical reaction with 5 % solution of trichloroacetic iron the urine of the child became olive green colour. Name the disease associated with the above symptoms.

A. PhenylketonuriaB. AlkaptonuriaC. TyrosinosisD. Albinism E. Maple syrup urine disease

Test 8After a long-term viral infection a 3-year-old child has frequent vomiting, loss of consciousness and convulsions. Medical examination revealed hyperammoniemia. These symptoms may be caused by ...

A. Increased putrefaction of proteins in large intestineB. Activation of amino acids decarboxylationC. Disorder of biogenic amines inactivation D. Disorder of ornithine cycleE. Inhibited activity of transamination enzymes

Test 9Deficiency of norepinephrine, serotonin and other biogenic amines in brain is considered tocause depression. Their concentration in the synapses may be increased by means of inhibition of ... activity.

A. Glutamate dehydrogenaseB. Diamine oxidaseC. L-amino acid oxidaseD. Monoamine oxidaseE. D-amino acid oxidase

Test 10Ammonia is well-known to be highly toxic chemical substance. The major mechanism of its detoxification in brain is ...

А. Biosynthesis of urea B. Formation of ammonia saltsC. Glutamine formation D. Transamination of amino acidsE.Reductive amination of α-amino acids

KEYS: 1 – D; 2 – B; 3 – A; 4 – E; 5 – C; 6 – C; 7 – A; 8 – D; 9 – D; 10 - C.

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CONTENTS

Pages

Preface 31. Enzymes 42. Vitamins 193. Energy Metabolism 304. Common Pathways of Catabolism 425. Metabolism of Carbohydrates 476. Metabolism of Lipids 707. Metabolism of Proteins 90

111

BIOCHEMISTRYIN SCHEMES

PART 1

Підписано до друку 08.11.2012 р. Формат 60х84/16.

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