REVIEW on CARBOHYDRATES

Aulanni’am

Biochemistry Laboratory_UB

CARBOHYDRATES

Hydrates of carbon [Cn(H2O)m] Polyhydroxyaldehyde or polyhydroxyketone, or

substance that gives these compounds on hydrolysis Most abundant organic compound in the plant world Chemically made up of skeletal C,H which is usually 2x

the number of C, highly variable number of O, occasional N & S

Linked to many lipids and proteins

FUNCTIONS of CARBOHYDRATES

Storehouses of chemical energy Glucose,starch, glycogen

Structural components for support Cellulose, chitin, GAGs

Essential components of nucleic acids D-ribose, 2-deoxy-D-ribose

Antigenic determinants Fucose, D-galactose, D-glucose, N-acetyl-D-

glucosamine, D-acetyl-D-galactosamine

SPECIFIC CARBOHYDRATESMonosaccharides

Glucose (dextrose, grape sugar, blood sugar) Can be stored as glycogen Most metabolically important monosaccharide

Fructose (levulose)

Galactose (brain sugar)

Mannose Targets lysosomal enzymes to their destinations Directs certain proteins from Golgi body to lysosomes

Disaccharides Sucrose (table sugar, cane sugar, saccharose)

glucose & fructose linked αβ1-2 Lactose (milk sugar) glu & gal linked β 1-4 Maltose (malt sugar) 2 glucose linked α 1-4 Trehalose (mycose) 2 glucose linked α 1-1 Gentiobiose (amygdalose) 2 glucose linked β 1-

6 Cellobiose 2 glucose linked β 1-4

CLASSES OF CARBOHYDRATESNumber of C

Triose, tetroses, pentose, hexose, heptulose

Number of saccharide units Monosaccharides, disaccharides, oligosaccharides (2 to 10

units), polysaccharides

Position of carbyonil (C=O) group Aldose if terminally located Ketose if centrally located

Reducing propertyReducing sugars (all monosaccharides)Nonreducing sugars (sucrose)

STRUCTURAL PROJECTIONS OF MONOSACCHARIDES

FISCHER by Emil Fischer (Nobel Prize in Chemistry 1902)

2-D representation for showing the configuration of a stereocenter

Horizontal lines project forward while vertical lines project towards the rear

D (R or +) or L (S or -)

HAWORTH by Walter Haworth

(Nobel Prize in Chemistry 1937)

A way to view furanose (5-membered ring) and pyranose (6-membered ring) forms of monosaccharides

The ring is drawn flat and viewed through its edge with the anomeric carbon on the the right and the oxygen atom on the rear

ANOMERIC CARBON

CHAIR & BOAT CONFORMATIONS

AMINO SUGARS

POLYSACCHARIDES STARCH

Storage carbohydrate in plants

Two principal parts are amylose (20-25%) & amylopectin (75-80%) which are completely hydrolyzed to D-glucose

• Amylose is composed of continuous, unbranched chain of 4000 D-glucose linked via α 1-4 bonds

• Amylopectin is a chain of 10,000 D-glucose units linked via α 1-4 bonds but branching of 24-30 glucose units is started via α 1-6 bonds

GLYCOGEN

Energy-reserve carbohydrate in animals

Highly branched containing approximately 106 glucose units linked via α 1-4 bonds & α 1-6 bonds

Well-nourished adult stores 350 g. of it equally divided between the liver and muscles

CELLULOSE Plant skeletal polysaccharide

Linear chain of 2200 glucose units linked via β 1-4 bonds

High mechanical strength is due to aligning of stiff fibers where hydroxyl form hydrogen bonding

ACIDIC POLYSACCHARIDES Also called mucopolysaccharides (MPS) or

glycosaminoglycans (GAG)

Polymers which contain carboxyl groups and/or sulfuric ester groups

Structural and functional importance in connective tissues

Interact with collagen to form loose or tight networks

ACIDIC POLYSACCHARIDES HYALURONIC ACID

Simplest GAG Contains 300-100,000 repeating units of D-glucuronic

acid and N-acetyl-D-glucosamine Abundant in embryonic tissues, synovial fluid, and the

vitreous humor to hold retina in place Joint lubricant & shock absorber

HEPARIN Heterogeneous mixture of variably sulfonated chains Stored in mast cells of the liver, lungs and the gut Naturally-occurring anticoagulant by acting as

antithrombin III and antithromboplastin Composed of two disaccharide repeating units A & B;

• A is L-iduronic acid-2-sulfate linked to 2-deoxy-2-sulfamido-D-galactose-6-sulfate

• B is D-glucuronic acid beta-linked to 2-deoxy-2-sulfamido-D-glucose-6-sulfate

HEPARAN SULFATE

CHONDROITIN SULFATE• Most abundant in mammalian tissues

• Found in skeletal and soft connective tissues

• Composed of repeating units of N-acetyl galactosamine sulfate linked beta1-4 to glucuronic acid

KERATAN SULFATE

DERMATAN SULFATE• Found in skin, blood vessels, heart valves, tendons, aorta,

spleen and brain

• The disaccharide repeating units are L-iduronic acid and N-acetylgalactosamine-4-sulfate with small amounts of D-glucuronic acid

GLYCOLYSIS The specific pathway by which the body gets

energy from monosaccharides

First stage is ACTIVATION At the expense of 2ATPs glucose is

phosphorylated

Step #1

formation of glucose-6-phosphate

Step # 2

isomerization to fructose-6-phosphate

Step # 3Second phosphate group is attached to yield fructose-1,6-

bisphosphate

Second stage is C6 to 2 molecules of C3

Step # 4Fructose-1,6-bisphosphate is broken down into two C3

fragments glyceraldehyde-3-phosphate (G-3-P) and

dihydroxyacetone phosphate (DHAP)

Only G-3-P is oxidized in glycolysis. DHAP is converted to G-3-P as the latter diminishes.

ATP-YIELDING Third stage Step # 5

Glyceraldehyde-3-phosphate is oxidized to 1,3-bisphosphoglycerate; hydrogen of aldehyde is removed by NAD+

Step # 6

Phosphate from the carboxyl group is transferred to the ADP yielding ATP and 3-phosphoglycerate

Step # 7

Isomerization of 3-phosphoglycerate to 2-phosphoglycerate

Step # 8 Dehydration of 2-phosphoglycerate to

phosphoenolpyruvate (PEP)

Step # 9Removal of the remaining phosphate to yield

ATP and pyruvate

Step # 10Reductive decarboxylation of pyruvate to

produce ethanol and CO2

REACTIONS OF GLYCOLYSIS

STEP REACTION ENZYME REACTION TYPE

ΔG in kJ/mol

1 Glucose + ATP G-6-P + ADP + H+

Hexokinase Phosphoryl transfer

-33.5

2 G-6-P F-6-P Phosphoglucose isomerase

Isomerization -2.5

3 F-6-P + ATP

F-1,6-BP + ADP + H+

Phosphofructo-kinase

Phosphoryl transfer

-22.2

STEP REACTION ENZYME REACTION TYPE ΔG in kJ/

mol

4 F-1,6-BP DHAP + GAP Aldolase Aldol cleavage -1.3

5 DHAP GAP Triose phosphate isomerase

Isomerization +2.5

6 GAP + Pi + NAD+ 1,3-BPG + NADH + H+

Glyceraldehyde-3-Phosphate Dehydrogenase

Phosphorylation coupled to oxidation

+2.5

7 1,3-BPG + ADP

3-phosphoglycerate +ATP

Phosphoglycer-ate kinase

Phosphoryl transfer

+1.3

8 3-phosphoglycerate 2-phosphoglycerate

Phosphoglyce-rate mutase

Phosphoryl shift +0.8

9 2-phosphoglycerate

PEP + HOH

Enolase Dehydration -3.3

10 PEP + ADP + H+ pyruvate + ATP Pyruvate kinase Phosphoryl transfer

-16.7

CITRIC ACID CYCLESTEP REACTION ENZYME PROSTHETI

C GROUPREACTION

TYPEΔGo

in kJ/

mol

1 acetylCoA + oxaloacetate + HOH

citrate + CoA + H+

Citrate synthase

Condensation -31.4

2a Citrate cis-aconitate + HOH Aconitase Fe-S Dehydration +8.4

2b Cis-Aconitate + HOH isocitrate Aconitase Fe-S Hydration -2.1

CITRIC ACID CYCLESTEP REACTION ENZYME PROSTHETI

C GROUPREACTION

TYPEΔGo

in kJ/

mol

3 Isocitrate + NAD+

α-ketoglutarate + CO2 +

NADH

Isocitrate

Dehydro-genase

Decarboxylation & oxidation

- 8.4

4 α-ketoglutarate + NAD+ CoA

succinyl CoA + CO2 +

NADH

α-ketogluta-rate dehydro-genase complex

Lipoic acid, FAD, TPP

Decarboxyla-tion & oxidation

-30.1

5 Succinyl CoA + Pi + GDP

succinate + GTP + CoA

Succinyl CoA synthet-ase

Substrate-level phosphoryla-tion

-3.3

CITRIC ACID CYCLESTEP REACTION ENZYME PROSTHETI

C GROUPREACTION

TYPEΔGo in

kJ/

mol

6 Succinate + FAD (enzyme-bound)

fumarate + FADH2

(enzyme-bound)

Succinate dehydro-genase

FAD, Fe-S Oxidation 0

7 Fumarate + HOH L-malate Fumarase Hydration -3.8

8 L-malate + NAD+ oxaloacetate + NADH + H+

Malate dehydro-genase

Oxidation +29.7

REGULATION OF TCA CYCLE Pyruvate

Acetyl CoA

Citrate

Isocitrate

Α-Ketoglutarate

Succinyl CoA

Succinate

Fumarate

Malate

Oxaloacetate

- ATP, acetyl CoA & NADH

- ATP & NADH

+ ADP

- ATP, succinyl CoA & NADH

Α-KGD

ICD

BIOSYNTHETIC ROLES

OF TCA CYCLE Pyruvate

Acetyl CoA

Citrate

Isocitrate

Α-Ketoglutarate

Succinyl CoA

Succinate

Fumarate

Malate

Oxaloacetate

Aspartate

Other amino acids,

purines & pyrimidines

Porphyrins, heme,

chlorophyll

Glutamate

Other amino

acids & purines

Fatty acids, sterols

NOTES TO REMEMBER

The unusual thing about the structure of N-acetylmuramic acid compared to other carbohydrates is the presence of a lactic acid side chain.

Cell walls of plants are cellulosic (polymer of D-glucose); bacterial cell walls consist mainly of polysaccharide crosslinked to peptide through murein bridges; and fungal cell walls are chitinous (polymer of N-acetyl-β-D-glucosamine)

Glycogen and starch differ mainly in the degree of chain branching.

Enantiomers are nonsuperimposable, mirror-image stereoisomers differing configuration on all carbons while diastereomers are nonsuperimposable nonmirror-image stereoisomers differing only on two carbons.

Fischer projection of glucose has 4 chiral centers while its Haworth projection has 5 chiral centers.

Sugar phosphate is an ester bond formed between a sugar hydroxyl and phosphoric acid.

A glycosidic bond is an acetal which can be hydrolyzed to regenerate the two original sugar hydroxyls.

A reducing sugar is one that has a free aldehyde group that can be easily oxidized.

Major biochemical roles of glycoproteins are signal transduction as hormones, recognition sites for external molecules in eukaryotic cell membranes, and defense as immunoglobulins.

L-sorbitol is made by reducing D-glucose.

Arabinose is a ribose epimer, thus, its derivatives ara-A and ara-C if substituted for ribose act as inhibitors in reactions of ribonucleosides.

Two best precursors for glycogen are glucose and fructose.

Cellulose because of the β- bonding is linear as to structure and structural as to role while starch because of α-bonding coils with energy storage role.

The highly branched nature of glycogen gives rise to a number of available glucose molecules at a time upon hydrolysis to provide energy. A linear one provides one glucose at a time.

The enzyme β-amylase is an exoglycosidase degrading polysaccharides from the ends. The enzyme α-amylase is an endoglycosidase cleaving internal glycosidic bonds.

Dietary fibers bind toxic substances in the gut and decreases the transit time, so harmful compounds such as carcinogens are removed from the body more quickly than would be the case with low-fiber diet.

The sugar portions of the blood group glycoproteins are the source of the antigenic difference.

Cross-linking can be expected to play a role in the structures of cellulose and chitin where mechanical strength is afforded by extensive hydrogen bonding.

Converting a sugar to an epimer requires inversion of configuration at a chiral center. This can only be done by breaking and reforming covalent bonds.

Vitamin C is a lactone (a cyclic ester) with a double bond between two of the ring carbons. The presence of a double bond makes it susceptible to air oxidation.

The sequence of monomers in a polysaccharide is not genetically coded and in this sense does not contain any information unlike the nucleotide sequence.

Glycosidic bonds can be formed between the side chain hydroxyls of serine or threonine residues and the sugar hydroxyls. In addition, there is a possibility of ester bonds forming between the side chain carboxyl groups of aspartate or glutamate and the sugar hydroxyls.

In glycolysis, reactions that require ATP are:

1. phosphorylation of glucose (HK,GK)

2. phosphorylation of fructose-6-phosphate (PFK)

Reactions that produce ATP are:

1. transfer of phosphate from 1,3-

bisphosphoglycerate to ADP (PGK)

2. transfer of phosphate from PEP to ADP (PK)

In glycolysis, reactions that require NADH are:1. reduction of pyruvate to lactate (LDH)2. reduction of acetaldehyde to ethanol

(alcohol dehydrogenase)Reactions that require NAD are:

1. oxidation of G-3-P to give 1,3-DPG (G-3-PD)

NADH-linked dehydrogenases are LDH, ADH & G-3-PD.

The purpose of the step that produces lactate is to reduce pyruvate so that NADH can be oxidized to NAD+ needed for the step catalyzed by glyceraldehyde-3-phosphate.

Aldolase catalyzes the reverse aldol condensation of fructose-1,6-bisphosphate to glyceraldehyde-3-phosphate and DHAP.

The energy released by all the reactions of glycolysis is 184.5 kJ mol glucose/mol. The energy released by glycolysis drives the phosphorylation of two ADP to ATP for each molecule of glucose, trapping 61.0 kJ mol/glucose. The estimate of 33% efficiency comes from the calculation (61.0/184.5) x 100 = 33%.

There is a net gain of two ATP molecules per glucose molecule consumed in glycolysis. The gross yield of 4 ATPs per glucose molecule, but the reactions of glycolysis require two ATP per glucose.

Pyruvate can be converted to lactate, ethanol or acetylCoA.

The free energy of hydrolysis of a substrate is the energetic driving force in substrate-level phosphorylation. An example is the conversion of glyceraldehyde-3-phosphate to 1,3-bisphosphoglycerate.

Coupled reactions in glycolysis are those reactions catalyzed by hexokinase, phosphofructokinase, glyceraldehyde-3-phosphate dehydrogenase, phosphoglycerokinase, and pyruvate kinase.

Isozymes allow for subtle control of the enzyme to respond to different cellular needs. For example, in the liver, LDH is most often used to convert lactate to pyruvate, but the reaction is often reversed in the muscles. Having a different isozyme in the liver and the muscle allows for those reactions to be optimized.

Fructose-1,6-bisphosphate can only undergo the reactions of glycolysis. The components of the pathway up to this point can have other metabolic fates.

The physiologically irreversible glycolytic steps are those catalyzed by HK, PFK and PK. Thus, they are controlling points in glycolysis.

Hexokinase is inhibited by glucose-6-phosphate. Phosphofructokinase is inhibited by ATP and citrate. Pyruvate kinase is inhibited is inhibited by ATP,

acetylCoA and alanine.

Phosphofructokinase is stimulated by AMP and fructose-2,6-bisphosphate.

Pyruvate kinase is stimulated by AMP and fructose-1,6-bisphosphate.

An isomerase is a general term for an enzyme that changes the form of a substrate without changing its empirical formula.

A mutase is an enzyme that moves a functional group such as a phosphate to a new location in a substrate molecule.

The glucokinase has a higher Km for glucose than hexokinase. Thus, under conditions of low glucose, the liver will not convert glucose to glucose-6-phosphate, using a substrate that is needed elsewhere. When the glucose concentration becomes higher, however, glucokinase will function to help phosphorylate glucose so that it can be stored as glycogen.

The net yield of ATP from glycolysis is the same, 2 ATP, when either fructose, mannose, and galactose is used. The energetics of the conversion of hexoses to pyruvate are the same regardless of hexose type.

The net yield of ATP is 3 from glucose derived from glycogen because the starting material is glucose-1-phosphate. One of the priming reactions is no longer used.

A reaction with a negative ΔGo is thermodynamically possible under standard conditions.

Individuals who lack the gene that directs the synthesis of the M form of the enzyme PFK can carry on glycolysis in their livers but suffer muscle weakness because they lack the enzyme in muscle.

The reaction of 2-PG to PEP is a dehydration (loss of water) rather than a redox reaction.

The hexokinase molecule changes shape drastically on binding to substrate, consistent with the induced fit theory of an enzyme adapting itself to its substrate.

ATP is an inhibitor of several steps of glycolysis as well as other catabolic pathways. The purpose of catabolic pathways is to produce energy, and high levels of ATP mean the cell already has sufficient energy. G-6-P inhibits HK and is an example of product inhibition. If G-6-P level is high, it may indicate that sufficient glucose is available from glycogen breakdown or that the subsequent enzymatic steps of glycolysis are going slowly. Either way there is no reason to produce more G-6-P.

Phosphofructokinase is inhibited by a special effector molecule, fructose-2,6-bisphosphate, whose levels are controlled by hormones. It is also inhibited by citrate, which indicates that there is sufficient energy from the TCA cycle probably from fat or amino acid catabolism.

PK is also inhibited by acetylCoA, the presence of which indicates that fatty acids are being used to generate energy for the citric acid cycle.

The main function of glycolysis is to feed carbon units to the TCA cycle. When these carbon skeletons can come from other sources, glycolysis is inhibited to spare glucose for other purposes.

Thiamine pyrophosphate (TPP) is a coenzyme in the transfer of 2-carbon units. It is required for catalysis by pyruvate decarboxylase in alcoholic fermentation. The important part of TPP is the five-membered ring where a C is found between an S and N. This carbon forms a carbanion and is extremely reactive, making it able to perform nucleophilic attack on carbonyl groups leading to decarboxylation of several compounds in different pathways.

TPP is a coenzyme required in the reaction catalyzed by pyruvate carboxylase. Because this reaction is a part of the metabolism of ethanol, less will be available to serve as a coenzyme in the reactions of other enzymes that require it.

Animals that have been run to death have accumulated large amounts of lactic acid in their muscle tissue, accounting for the sour taste of their meat.

Conversion of glucose to lactate rather than pyruvate recycles NADH.

The formation of fructose-1,6-bisphosphate is the committed step in glycolysis. It is also one of the energy-requiring steps of the said pathway.

A positive ΔGo does not necessarily mean that the reaction has a positive ΔG. Substrate concentrations can make a negative ΔG out of a positive ΔGo.

The entire pathway can be looked at as a large coupled reaction. Thus, if the overall pathway has a negative ΔG, an individual step may be able to have a positive ΔG and the pathway can still continue.

In glycogen storage, the reactions that require ATP are:1. formation of UDP-glucose from glucose-1-

phosphate and UTP (indirect requirement since ATP is needed to regenerate UTP) (UDP-glucose phosphorylase)

2. regeneration of UTP (nucleoside phosphate kinase)3. carboxylation of pyruvate to oxaloacetate (pyruvate

carboxylase)Reactions that produce ATP are NONE.

Three differences between NADPH and NADH1. phosphate at 2’ position of ribose in NADPH2. NADH is produced in oxidative reactions that yield

ATP while NADPH is a reducing agent in biosynthesis.

3. Different enzymes use NADH as a coenzyme compared to those that require NADPH.

In glycogen storage, there is no reaction that requires acetylCoA but biotin is required in the carboxylation of pyruvate to oxaloacetate.

The four fates of glucose-6-phosphate are:• Converted to glucose (gluconeogenesis)• Converted to glycogen (glycogenesis)• Converted to pentose phosphates• Hydrolyzed to pyruvate (glycolysis)

In making equal amounts of NADPH and pentose phosphates, it only involves oxidative reactions. In making mostly or purely NADPH, the use of oxidative reactions, transketolase and transaldolase reactions, and gluconeogenesis are required. In making mostly or only pentose phosphates, needed reactions are transketolase, transaldolase, and glycolysis in reverse.

Transketolase catalyzes the transfer of 2-carbon unit, whereas transaldolase catalyzes the transfer of a 3-carbon unit.

It is essential that the mechanisms that activate glycogen synthesis also deactivate glycogen phosphorylase because they both occur in the same cell compartment. If both are on at the same time, a futile ATP hydrolysis results. On/off mechanism is highly efficient in its control.

UDPG, in glycogen biosynthesis, transfers glucose to the growing glycogen molecule.

Glycogen synthase is subject to covalent modification and to allosteric control. The enzyme is active in its phosphorylated form and inactive when dephosphorylated.

AMP is an allosteric inhibitor of glycogen synthase, whereas ATP and glucose-6-phosphate are allosteric activators.

In gluconeogenesis, biotin is the molecule to which carbon dioxide is attached to the process of being transferred to pyruvate. The reaction produces oxaloacetate, which then undergoes further reactions of gluconeogenesis. Biotin is not used in glycogenesis and PPP.

In gluconeogenesis, glucose-6-phosphate is dephosphorylated to glucose (last step); in glycolysis, G-6-P isomerizes to fructose-6-phosphate (early step).

The Cori cycle is a pathway in which there is cycling of glucose due to glycolysis in muscle and gluconeogenesis in liver. The blood transports lactate from muscle to liver and glucose from liver to muscle.

There is a net gain of 3, rather than 2, ATP when glycogen, not glucose, is the starting material of glycolysis.

Control mechanisms are important in metabolism. They are: Allosteric control (takes place in msec) Covalent control (takes place from s to min) Genetic control ( longer time scale)

Enzymes, like all catalysts, speed up the forward and reverse reaction to the same extent. Having different catalysts is the only way to ensure independent control over the rates of the forward and the reverse process.

The glycogen synthase is an exergonic reaction overall because it is coupled to phosphate ester hydrolysis.

Increasing the level of ATP is favorable to both gluconeogenesis and glycogen synthesis.

Decreasing the level of fructose-1,6-bisphosphate would tend to stimulate glycolysis, rather than gluconeogenesis and glycogen synthesis.

If a cell needs NADPH, all the reactions of the PPP take place. If a cell needs ribose-5-phosphate, the oxidative portion of the pathway can be bypassed and only the nonoxidative reshuffling reactions take place. The PPP does not have a significant effect on the ATP supply of a cell.

Glucose-6-phosphate is expectedly oxidized to a lactone rather than an open-chain ester because the latter is easy to hydrolyze.

In the PPP resshuffling reactions, without an isomerase, all the sugars involved are keto sugars that are not substrates for transaldolase.

Sugar nucleotides (UDPG) have two phosphates which when hydrolyzed drives towards the polymerization of glycogen. Thus, they are fit for glycogenesis.