chapter 7 catabolism of proteins. nutritional function of proteins functions: structural catalytic,...

Chapter 7 Catabolism of Proteins

Nutritional Function of Proteins Functions: Structural

Catalytic,

Transport action

Signaling and hormonal functions

Source of energy (16.7kJ/g)

Nutritional Requirement of Proteins Nitrogen Balance

Proteins contain about 16% nitrogen

Intake N = losses N

Intake N > Losses N

Intake N < Losses N

Nutritional Quality of Proteins Essential Amino Acids

cannot be synthesized by the body and must be obtained from diet

Eight nutritional essential amino acids Tryptophan

Phenylalanine

Lysine

Threonine

Valine

Leucine

Isoleucine

methionine

Nutritional Quality of Proteins Non-essential amino acids

synthesized in the body

synthesized by the transamination of a-keto acids

Tyrosine and cysteine

synthesized in the body by using essential amino acids

from phenylalanine and methionine respectively

semi-essential

Digestion of Dietary Proteins

Dietary proteins are digested in the stomach and intestine

Digestion of Protein in the Stomach

The digestion of protein. Protein is broken down into amino acids by the enzymes pepsin (secreted by the stomach) and trypsin and peptidase (in the small intestine).

Table 1. Phases of Digestion and Absorption of Protein and its Degradative Products

Phase of Digestion

Location Agents Outcome

5. Cleavage of di-/tripeptides

transport to capillaries

epithelial cell – cytoplasm

contraluminal membrane

dipeptidasestripeptidases

facilitated diffusion

free amino acids from di-/tripeptides;

amino acids transported into capillaries

4. Absorption intestinal epithelial cell brush border membrane

transport systems uptake into epithelial cell

3. Brush Border Surface

brush border surface of intestine

endopeptidases and aminopeptidases

free amino acids anddi-/ tripeptides

2. Pancreatic Proteases

lumen of small Intestine

trypsin, chymotrypsin,elastase, and carboxypeptidases

free amino acids andoligopeptides – 2 to 8 amino acids

1. Gastric Digestion

stomach stomach acid

pepsin

denaturation

large peptide fragments + some free amino acids

Gastric Parietal CellPlasma

Lumen of the Stomach

CO2

HCO3-

H+

ATP

ADP + Pi

K+

H+

CO2 +H2O

carbonic anhydrase

H2CO3

HCO3-

Cl-Cl- Cl-

Production of gastric acid and its secretion

H+,K+-ATPase

Phase 1- Gastric digestion

DietaryProtein

denaturation by stomach acid

Figure 2. Gastric digestion of dietary protein.

large peptide fragmentsfree amino acids

hydrolysis by pepsin

Pyloric sphincter

Duodenum

Gastric Chief Cells

Acid from parietal cells denatures protein to be more susceptible to pepsin cleavage .

Pepsinogen activated to pepsin by autoactivation and autocatalysis by pepsin.

Large peptide fragments/some amino acids pass through the pyloric sphincter to the duodenum

Pepsinogen

autocatalysis

Pepsin

autoactivation (intramolecular cleavage)

aaaa

aaaa

PancreaticAcinar

Cell

DuodenalEndocrineCell

Blood-stream

MucosalEpithelialCells

Entero-peptidase

DuodenalEndocrineCell

Phase 2- Digestion by pancreatic proteases

Trypsinogen

Trypsin

free amino acids from gastric digestion

CCK-PZCCK-PZ

(hydrolysis)

Figure 3. Secretion, activation and action of pancreatic proteases and brush border endopeptidases and aminopeptidases

DuodenalEndocrineCell CCK-PZ

PancreaticAcinar

Cell

Blood-stream


Entero-peptidase(hydrolysis)



Trypsinogen

Trypsin


HCO3-

neutralizes acid

Secretin

autocatalysis


DuodenalEndocrineCell CCK-PZ Secretin

PancreaticAcinar

Cell

Blood-stream





Trypsinogen

Trypsin

autocatalysis

ChymotrypsinogenProelastaseProcarboxypeptidases

ChymotrypsinElastaseCarboxypeptidases

catalysis


HCO3-

neutralizes acid



DuodenalEndocrineCell CCK-PZ Secretin

PancreaticAcinar

Cell

Blood-stream




Phase 2- Digestion by pancreatic proteasesPhase 3- Digestion at the brush border

Trypsinogen

Trypsin

autocatalysis

ChymotrypsinogenProelastaseProcarboxypeptidases

ChymotrypsinElastaseCarboxypeptidases

catalysis

amino acidsdipeptidestripeptides


HCO3-

neutralizes acid

brush border endo-/aminopeptidases hydrolyze products; amino acids, di-/tripeptides absorbed by epithelial cells

. Summary of the gastric and pancreatic digestive proteases

Protease Source Proteasefamily

Proenzyme Activation Specificity

Trypsin (endo-) pancreas serine trypsinogen enteropeptidasetrypsin

basic (arg, lys)

Chymotrypsin(endo-)

pancreas serine chymo-trypsinogen

trypsin bulky aromatic(trp, phe, tyr, met)

Elastase(endo-)

pancreas serine proelastase trypsin small neutral R groups(gly, ser, ala)

Carboxypeptidase A (exo-)

pancreas zinc procarboxy-peptidase A

trypsin aromatic(tyr, phe, trp)hydrophobic(val, leu, ile)

Carboxypeptidase B (exo-)

pancreas zinc procarboxy-peptidase B

trypsin basic(arg, lys)

Pepsin (endo-) stomach aspartate pepsinogen autoactivation/H+; pepsin

aromatic (tyr,phe, trp)acidic (glu)

LUMEN OF INTESTINE

Intestinal Epithelium

Amino acids Di-, tri-

peptides Na+

= Na+-dependent co-transport


Brush border

Amino acids

Dipeptides, tripeptides

Phase 4 - Absorption

Figure 4. Absorption of amino acids and di- and tripeptides from the intestinal lumen

3Na+

2K+ ATP

ADP + Pi

= Na+,K+-ATPase

3Na+2K+

Na+

Dipeptidases,

tripeptidases

a) neutral amino acids (uncharged aliphatic and aromatic)

b) basic amino acids and cystine (Cys-Cys)

c) acidic amino acids (Asp, Glu)

d) imino acids (Pro)

e) dipeptides and tripeptides

BRUSH BORDER TRANSPORT SYSTEMS

Figure 4. Absorption of amino acids and di- and tripeptides from the intestinal lumen

LUMEN OF INTESTINE

Intestinal Epithelium

Amino acids Di-, tripeptides

Na+

= facilitated diffusion

= Na+-dependent co-transport


Amino acids

Brush border

Dipeptides, tripeptides

Dipeptidases, tripeptidases

Phase 4 - Absorption

Phase 5

capillaries

Phase 5

3Na+

2K+ ATP

ADP + Pi

= Na+,K+-ATPase

3Na+2K+

Putrefaction Decomposition of amino acids and proteins by

bacteria Most ingested proteins are absorbed from the

small intestine 95% of total dietary proteins

Undigested proteins

pass into the large intestine

Bacterial activity occurs

Putrefaction

Bacteria putrefaction produces some nutritional benefits,

Vitamin K, Vitamin B12, Folic acid

Toxic for human Amines, phenol, indole, H2S

Production of Amines Production of phenol Production indole Production of H2S Production of Ammonia

Page 209

Degradation of Protein in Cells

The half-life of proteins is determined by rates of synthesis and degradation

A given protein is synthesized at a constant rate KS

A constant fraction of active molecules are destroyed per unit time

C is the amount of Protein at any time

KD is the first order rate constant of enzyme degradation, i.e., the fraction destroyed per unit time, also depends on the particular protein

KS is the rate constant for protein synthesis; will vary depending on the particular protein

Rate of Turnover = dC

dt = KS - KDC

Steady-state is achieved when the amount of protein synthesized per unit time equals the amount being destroyed

dC

dt= 0 KDC = KS t 1/2 =

0.693

KD

Proteinconcentration(enzyme activity)

Hours after stopping synthesis

C

Stop protein synthesis,measure rate of decay

Steps in Protein Degradation

Transformation to a degradable form(Metal oxidized, Ubiquination, N-terminal residues, PEST sequences)

Lysosomal DigestionLysosomal Digestion 26S Proteasome digestion26S Proteasome digestion

Proteolysis to peptides

UbiquinationUbiquination

ATP

AMP + PPi

KFERQ8 residue fragments

7 type, 7 type subunits

N-end rule: DRLKF: 2-3 min AGMSV: > 20 hrPEST: Rapid degradation

COO-Ubiquitin

Glycine at C terminal of Ubiquitin

SC

O

E1

HSATP

AMP + PPi

E1

HS

HS E1

E2H3N+

NH3+

NH3+

N

NCO

N CO

CO

ATP

AMP + PPi

Ubiquitin-specific proteases(26S proteasome)

Degradedprotein + Ubiquitin

Ubiquination

Ubiquitin activating enzyme

Activationof Ubiquitin

Ubiquitin conjugating enzyme 20 or more per cell

SC

O

E23

E2 SH3

Ubiquitin ligase

E3

Page 211

CPoly Ubiquitin

NH

O

Amino Acid Catabolism Deamination of Amino Acids

removal of the a-amino acids

Oxidative Deamination

Non-oxidative Deamination

Transamination

Oxidative Deamination

Only a few amino acids can be deaminated directly. Glutamate Dehydrogenase catalyzes a major reaction that effects net removal of N from the amino acid pool . Glutamate Dehydrogenase is one of the few enzymes that can utilize either NAD+ or NADP+ as electron acceptor. Oxidation at the -carbon is followed by hydrolysis, releasing NH4

+.

At right is summarized the role of transaminases in funneling amino N to glutamate, which is deaminated via Glutamate Dehydrogenase, producing NH4

+.

Non-oxidative Deamination Serine Dehydratase catalyzes:

serine pyruvate + NH4

Transamination

Transaminase enzymes (aminotransferases) catalyze the reversible transfer of an amino group between two -keto acids.

An

Example of a transaminase reaction is shown at right.

•Aspartate donates its amino group, becoming the -keto acid oxaloacetate. -Ketoglutarate accepts the amino group, becoming the amino acid glutamate.

In another example shown at right, alanine becomes pyruvate as the amino group is transferred to -ketoglutarate.

Transaminases equilibrate amino groups among available -keto acids. This permits synthesis of non-essential amino acids, using amino groups derived from other amino acids and carbon skeletons synthesized in the cell. Thus a balance of different amino acids is maintained, as proteins of varied amino acid contents are synthesized.

Mechanism of Transamination

The prosthetic group of the transaminase enzyme is pyridoxal phosphate (PLP), a derivative of vitamin B6.

In the "resting" state, the aldehyde group of pyridoxal phosphate is in a Schiff base linkage to the -amino group of an enzyme lysine residue.

The -amino group of a substrate amino acid displaces the enzyme lysine, to form a Schiff base linkage to PLP.

The active site lysine extracts a proton, promoting tautomerization (shift of the double bond), followed by reprotonation with hydrolysis.

What was an amino acid leaves as an -keto acid. The amino group remains on what is now pyridoxamine Phosphate (PMP).

A different -keto acid reacts with PMP, and the process reverses, to complete the reaction.

Purine Nucleotide Cycle The activity of L-glutamate dehydrogenase

is low in the skeletal muscle and heart. In this tissues purine nucleotide cycle

Figure 9-7 page 216

Metabolism of One Carbon Units

One carbon units are one carbon containing groups produced in catabolism of some amino acids.

Methyl (-CH3), methylene (=CH2), formyl (O=CH-) and formimino (HN=CH-)

tetrahydrofolate (FH4) One carbon units are carried by

tetrahydrofolate (FH4), a reduced form of folic acid.

tetrahydrofolate (FH4) FH4 is formed in

reduction of folic acid catalyzed by dihydrofolate reductase. The four hydrogens are added to the four atoms of folic acid in positions 5 to 8. The N5 and N10 nitrogen atoms of FH4 participate in the transfer of one carbon groups

Production of One Carbon Units

Either glycine or serine can act as methylene donor, giving N5,N10-methyleneTHF. This behaves as "virtual formaldehyde" H2C=O in

reactions. The oxidation level can be changed to methyl or methenyl by reduction or oxidation; methenylTHF can be hydrolyzed to formylTHF.

Production of One Carbon Units from Histidine N5-formimino-

tetrahydrofolate, produced in the pathway for degradation of histidine

In the pathway of histidine degradation, conversion of N-formiminoglutamate to glutamate involves transfer of the formimino group to tetrahydrofolate (THF), yielding N5-formimino-THF.

Adenosylmethionine (SAM)

S-adenosylmethionin (SAM) is the major donor of methyl group. FH4 can carry a methyl group on its N5 atom, but its transfer potential is too low for most biosynthetic methylation.

The activated methyl donor is SAM, which is synthesized by the transfer of an adenosyl group from ATP to the sulfer atom of methionine. The S-adenosylhomocysteine is formed when the methyl group of SAM is transferred to an acceptor.

Conversion of One Carbon UnitsFigure 9-13

Metabolism of Methionine, Cysteine and Cystine

Sulfur-containing amino acids Methionine is an essential amino acid

Methionine cycle and methylation

In methionine cycle, the adenosyl group of ATP is transferred to a sulfur atom of methionine by methionine adenosyltransferase to form S-adenosylmethionine (Sam)


All phosphates of ATP are lost in this reaction. The sulfonium ion of methionine is highly reactive and the methyl group of SAM is good leaving group. SAM then transfers the methyl group to some acceptors for their methylation by methyltransferase.


The resulting S-adenosylhomocysteine is cleaved by adenosylhomocysteinase to produce homocysteine and adenosine.

Homocysteine accepts a methyl group from N5-methyl-FH4 to regenerate methionine.


This reaction is catalyzed by homocysteine methyltransferase, which requires vitamin B12 as a cofactor. This is the only reaction known that uses methyl-FH4 as a methyl group donor.

The net result of the reaction is donation of a methyl group and regeneration of methionine to complete the methionine cycle.


Person with elevated serum levels of homocysteine have a high risk for coronary heart disease and arteriosclerosis. The molecule basis of the action of homocysteine has not been clearly identified. It appears to damage cells of blood vessels and to increase the growth of vascular smooth muscle. Treatment with vitamin B12, folic acid and vitamin B6 is effective in reducing homocysteine level in some people.

Creatine and Creatine Phosphate

Glycine, areginine and methionine participate in synthesis of creatine

Transfer of guanidine group from arginine to glycine forms guanidoacetate catalyzed by transamidinase in kidney

Creatine and Creatine Phosphate Synthesis of creatine is

completed by methylation f guanidoacetate in the liver. This reaction is catalyzed by guanidoacetate methyltransferase.

SAM serves as a donor of a methyl group.

Storage of “high energy” phosphate from ATP, creatine converts to creatine phosphate particularly in cardiac and skeletal muscle catalyzed by creatine kinase (CK)

Creatine and Creatine Phosphate

This reaction is reversible and creatine phosphate can readily convert ADP to ATP in muscle to meet the energy requirement. The amount of creatine in the body is related to muscle mass.

Creatinine is derived from dephosphorylation of creatine phosphate and also formed by hydrolysis of creatine nonenzymatically.

Creatinine has no function and is excreted in urine. The amount of creatinine eliminated by an individual is constantly from day to day. When a 24 hours urine sample is requested, the amount of creatinine in sample can be used as a gross determining test to know renal function.

Cysteine and Cystine Conversion of Cystein

e To Cystine two molecules of cystein

e are linked by a disulfide bond to form cystine. The major catabolic pathway of cystine is conversion of cysteine catalyzed by cystine reductase. The disulfide bond of cystine is important to maintain the conformation and function of proteins

Synthesis of Taurine Cysteine is the precusor of taurine. The

major oxidative metabolite of cysteine is cysteine sulfinate, which is further decarboxylation to form taurine.

Taurine is found rich in brain. It appears to play role in brain development, but its exact role is unknown

Figure. Page 229

Formation 3’-phosphoadenosine 5’phosphosulfate (PAPS)

Sulfate is produced mostly from metabolism of cysteine. Catabolism of cysteine produces pyruvate, NH3 and H2S. Oxidation of H2S forms sulfate. Some sulfate group for addition to biomolecules, such as in biosynthesis of chondroitin sulfates and keratan sulfate.

Figure. Page 229

Glutathione Glutathione is the tri

peptide Gamma-glutamylcysteinylglycine containing a sulfhydryl group. Glutathione has several important role.

serves as a transporter in the gamma-glutamyl cycle for amino acids across cell membranes

protects erythrocytes from oxidative damage

Glutathione cycles (Meister cycle)

figure.9-16

The enzyme gamma-glutamyl transpeptidase, located on the cell membrane of kidneys and other tissue cells, catalyzes glutathion (GSH) to transfer its glutamyl group to amino acid, then the gamma-glutamyl-ammino acid is transported inside of the cell.


figure.9-16

The gamma-glutamyl-amino acid releases amino acid and 5-oxiproline. This is the process for amino acid transportation into the cell.

The 5-oxiproline converts to glutamate under the action of enzyme and uses

ATP.


figure.9-16 The 5-oxiproline converts to

glutamate under the action of enzyme and uses ATP.

Glutamate and the other parts of GSH, glycine and cysteine, are regenerated GSH in cytosol and 2 ATPs are used. So 3 ATPs are required for the transportation of each amino acid.

The key enzyme of the gamma-glutamyl cycle is gamma-glutamyl transpeptidase which is found in high levels in the kidneys


figure.9-16 Glutathion cycles between a

reduced form with a sulfhydryl group (GSH) and an oxidized form (GSSG), in which two GSHs are linked by a disulfide bond. GSH is reductant, its sulhydryl group can be used to reduce peroxides formed during oxygen transport.

Glutathione plays a key role in detoxification by acting with hydrogen peroxide and organic peroxide.

Glutathion peroxidase catalyzes this reaction, in which GSH converts to GSSG. Then GSSG is reduced to GSH by glutathione reductase, an enzyme containing NADPH as a cofactor.

Metabolism of Aromatic Amino Acids Formation of Tyrosine

from phenylalanine First product in

degradation of phenylalanine

Metabolism of Aromatic Amino Acids Formation of

Tyrosine from phenylalanine

first product in degradation of phenylalanine

Phenylalanine hydroxylase

Phenylketonuria (PKU) Small amounts of

phenylalanine can convert to phenylpyruvate by transamination to remove an amino group in a healthy person.

If a genetic deficiency of phenylalanine hydroxylase occurs, phenylketonuria is caused Phenylalanine

hydroxylase

Phenylketonuria (PKU) PKU is the most common autosomal disease. Over 170

mutations in the gene have been reported. The elevated phenylpyruvate, phenyllacetate (reduction product of phenylpyruvate) and phenylacetate (decarboxylation of phenlpyruvate) excreted in urine give urine its characteristic odor. The neurological symptoms and light color of skin and eyes are generally toxic effects of high levels of phenylpyruvate and low concentrations of tyrosine. The conventional treatment is to feed the effected infant a diet low in phenylalanine with dietary protein restrictions.

Figure 9-17 Metabolism and major derivatives of phenylalanine and tyrosine

Metabolism of Tyrosine

The first step in catabolism of tyrosine is transamination catalyzed by tyrosine transaminase to produce p-hydroxyphenylpyruvate, which converts to homogentisate by oxidase. Homogentisate is then cleaved to fumarate and acetoacetate. Fumarate is used in the TCA cycle for energy or for gluconeogenesis. Acetoacetate can convert to acetyl CoA for lipid synthesis or energy.

Production of Dopamine, Epinephrine and Norepinephrine

Some tyrosine is used as a precursor of catecholamines (term of dopamine, epinephrine and norepinephrine)

The first step in the synthesis of catecholamines is catalyzed by tyrosine hydroxylase, which is an enzyme dependent on tetrahydrobiopterin.

The product of this reaction is dihydroxyphenylalanin, known as Dopa. A product of decarboxylation of Dopa is dopamine, which is a neurotransmiter. Parkinson’s disease is induced by decreasing production of dopamin.

The adrenal medulla converts dopamine to norepinephrine by dopamine hydroxylase, which accepts a methyl group from S-adenosylmethionine to form epinephrine.

Synthesis of MelaninFigure 9-17

Tyrosine is precursor of melanin. Dopa is the intermediate in the synthesis of both melanin and epinephrine.

Different enzymes dydroxylate tyrosines in melanocytes and other cell type. In pigment cell, tyrosine is hydroxylated to form Dopa by tyrosinase, a copper-containing enzyme.

Dopa forms dopamine then converts it to indo-5-6-quinone. Melanin is polymers of these tyrosine catabolites with proteins from the eyes and skin. There are various types of melanin, which are all aromatic quintines complexes giving color, colorless, yellow and dark to the skin.

Albinism

Albinism results from a genetic lack of tyrosinase. Lack of pigment in the skin makes a patient sensitive to sunlight and increases the incidence of skin cancer in addition to burns. Lack of pigment in eyes may induce photophobia

Production of Thyroid Hormone

Tyrosine is the precursor of the thyroid hormone: T4 and T3. The thyroid hormone has importance in regulating the general metabolism, development and tissue differentiation. Iodination of tyrosine residues in thyroglobulin forms T4 and T3

tetraiodothyronine, T4:

:

triiodothyronine,T3.

Metabolism of TryptophanFigure 9-18

Metabolism of TryptophanFigure 9-18

Trytophan

precursor of nicotinic acid, one of the B vitamins.

b hydroxylation and decarboxylation forms 5-hydroxytryptamine (5-HT, serotonin)

Melatonin is a derivative of tryptophan, N-acetyl-5-methoxytryptamine. It is a sleep-inducing molecule and is synthesized in the pineal gland and retina mostly at night. Melatonin appears to function by inhibiting synthesis and secretion of other neurotransmitters, such as dopamine and GABA.

Degradation of Branched-Chain Amino Acids (BCAAs)

Figure 9-19

Valine, isoleucine and leucine are branched-chain amino acids (BCCAs).

BCAAs transaminases are present at a much higher level in muscle than that in liver

Valine converts to succinyl CoA. So it is a glucogenic amino acid. Leucine converts to acetyl CoA and acetoacetate. Leucine is a ketogenic amino acid. Isoleucine produces acetyl CoA and succinyl CoA and is both glycogenic and ketogenic amino acid. All these intermediates of BCAAs degradation are oxidation in the TCA cycle to support energy in muscle.

Transport of Ammonia in Blood At physiological pH, 98.5% exists as

ammonium ion (NH+4) Only traces of NH3 are present Even trace of NH3 are toxic to the nervous

system NH3 is rapidly removed

Glutamine synthetase fixes ammonia as glutamine

Formation of glutamine is catalyzed by glutamine synthetase. Synthesis of the amide bond of glutamine is accomplished at the expense of hydrolysis of one mole of ATP to ADP and Pi.

Glutamine Synthetase

Hydrolysis of glutamine produces glutamate and NH3 in the liver and kidneys

Glutamine supports an amide group for synthesis of asparagine from aspartate by asparagine synthetase. Since certain tumors such as leukemic cells seem to lose this ability and exhibit abnormally high requirements for asparagine and glutamine, hydrolysis of asparagine is catalyzed by asparaginase. So, exogenous asparaginase and glutaminase had been tested as antitumor age

nts

Alanine-glucose cycleFigure 9-8

Muscles generate over half of the total metabolism pool of amino acids. The ammonia produced in catabolism of amino acids in muscle is accepted by pyruvate to form alanine, which is released into the blood.

Alanine appears to be the vehicle of ammonia for transport in the blood

The liver takes up the alanine and converts it back into pyruvate by transamination

The resulting pyruvate can be converted to glucose by the gluconeogenesis pathway and an amino group eventually appears as urea.

Glucose formed in gluconeogenesis is released into the blood and taken up by muscles.

Glycolysis of glucose produces pyruvate, which is then resynthesized alanine. This is called alanine-glucose cycle

Formation of Urea (Urea Cycle)

Urea Cycle

The urea cycle takes place partly in the cytosol and partly in the mitochondria, and the individual reactions are as follows

Urea Cycle carbamyl phosphate synthetase 1 [CPS1] This liver mitochondrial enzyme converts the ammonia

produced by glutamate dehydrogenase into carbamyl phosphate (=carbamoyl phosphate) which is an unstable high energy compound. It is the mixed acid anhydride of carbamic acid and phosphoric acid, and requires two

molecules of ATP to drive its synthesis.

Urea Cycle

CPS1 is an allosteric enzyme and is absolutely dependent up on N-acetylglutamic acid for it activity

Urea Cycle CPS1 deficiency results in hyperammonemia. The neonatal

cases are usually lethal, but there is also a less severe, delayed-onset form. Ammonia-dependent CPS1 is present only in the liver mitochondrial matrix space. It should be distinguished from a second cytosolic glutamine-dependent carbamyl phosphate synthetase [CPS2] which is found in all tissues and is involved in pyrimidine biosynthesis. Carbamyl phosphate synthesis is a major burden for liver mitochondria. This enzyme accounts for about 20% of the total protein in the matrix space. Glutamate dehydrogenase is also present in very large amounts.

Urea Cycle The next reaction also takes place in the liver

mitochondrial matrix space, where ornithine is converted into citrulline

ornithine transcarbamylase [OTCase]

Urea Cycle Citrulline is transported out of the mitochondria into

cytosol by the mitochondrial inner membrane transport system. Once in the cytosol, citrulline condenses with aspartate and the reaction is driven by ATP. In this way aspartate contributes the second nitrogen atom to urea, the first having come from glutamate

Urea Cycle

Production of arginino-succinate is an energetically expensive process, since the ATP is split to AMP and pyrophosphate. The pyrophosphate is then cleaved to inorganic phosphate using pyrophosphatase, so the overall reaction costs two equivalents of high energy phosphate per mole.

Urea Cycle Elimination of fumarate from arginino-

succinate then yields arginine.

arginino-succinate lyase

Urea Cycle

Fumarate can be converted into oxaloacetate under catalysis of some enzymes as in the TCA cycle. Oxaloacetate can be converted to aspartate by transamination. The aspartate is then reutilized in the urea cycle

Urea Cycle Cleavage of arginine by arginase to produce urea

regenerates ornithine, which is then available for another round of the cycle.

Urea Cycle

Since humans can not metabolize urea, it is transported to the kidneys for excretion. Some urea that enters the intestinal tract is cleaved by bacteria urease, the resulting ammonia being absorbed and treated by the liver

Note that of the two nitrogen atoms of urea, one comes from carbamoyl phosphate, being ultimately derived from ammonia. The other nitrogen is derived from the a-amino group of aspartate which in turn is obtained from transamination of oxaloacetate with glutamate. The formation of one molecule of urea requires the hydrolysis of four high-energy phosphate groups from 3 molecules of ATP

The overall reaction is as follows:2NH3 + CO2 +3ATP + 3H2O -> H2N-CO-NH2 +2ADP + AMP +4Pi

Urea Cycle (review)1. Occurs in the liver mitochondria and cytosol

2. Starts with carbamoyl-PO4

3. Ends with arginine

4. Requires aspartate

5. Requires 3 ATPs to make one urea

NH4+ + HCO3

- + 2 ATP

Synthesis of Carbamoyl-PO4

H2NC

O

O-P-O

O

O

~

High energy bond

+ 2 ADP + Pi

Carbamoyl phosphate Synthetase ICarbamoyl phosphate Synthetase I

Ornithine

Citrulline

Argininosuccinate

Arginine

Carbamoyl-PO4

Aspartate

Urea

ATP

Urea Cycle

Urea Cycle

H2ONH

CH2

CH2

CH2

COO-

CH3N

H

H2N=CNH2+

NH3

CH2

CH2

CH2

COO-

CH3N

H

+

C

H2N NH2

O

COPO3H2N

O

COO-

CH2

H3N+-C-H

NH3

CH2

CH2

+

+

O=C

COO-

CH2

H3N+-C-H

NH

CH2

CH2

NH2

+ OPO3=

Ornithine

Carbamoyl-PO4Citruline

Reactions of Urea Cycle

O=C

COO-

CH2

H3N+-C-H

NH

CH2

CH2

NH2

+

COO-

CH2

COO-

H-C-NH3

+

=C

COO-

CH2

H3N+-C-H

NH

CH2

CH2

NH2

COO-

CH2

COO-

H-C-NL-Aspartate

Argininosuccinate

ATP ADP + Pi

Mitochondria

Cytosol

=C

COO-

CH2

H3N+-C-H

NH

CH2

CH2

NH2

COO-

CH2

COO-

H-C-N =C

COO-

CH2

H3N+-C-H

NH

CH2

CH2

NH2

H2N+

COO-

COO-

CH2

C-OHH

+

COO-

COO-

C

C

H

H

COO-

COO-

CH2

C=O

COO-

CH2

COO-

H-C-NH3

+

Fumarate

L-MalateOxaloacetateL-Aspartate

Cytosol

L-Arginine

=C

COO-

CH2

H3N+-C-H

NH

CH2

CH2

NH2

H2N+

COO-

CH2

H3N+-C-H

NH3

CH2

CH2

+

H2NC

O

NH2

Urea

+

Ornithine

L-Arginine

H2O

Return to Mitochondria

Nitric Oxide Arginine also serves as a direct precursor of nitric oxide (NO). The

free-radical gas NO is the potent muscle relaxant and short-lived signal molecule. Nitric oxide is formed by the catalysis of the cytosol enzyme nitric oxide synthase (NOS), which is a very complex enzyme with five cofactors: NADPH, FAD, FMN, heme and tetrahydrobiopterin.

The substrate in the reaction is arginine and products are citrulline and NO. Oxygen is required in the complex reaction. NO plays an important role in many physiologic and pathologic processes

Decarboxylation of Amino Acids

Decarboxylation of amino acids forms amine. This reaction is catalyzed by decarboxylase, which contains pyridoxal phosphate as a cofactor. Amines always have potential physiological effects.

GABA gamma-Aminobutyric acid (GABA) is formed by

pyridoxal phosphate-dependent enzyme, L-glutamate decarboxylase, which is principally present in brain tissue. GABA functions as inhibitory neurotransmitter. GABA, catalyzed by gamma-aminobutyrate transaminase, forms succinate and semialdehyde, which may be oxidized to form succinate and via TCA cycle to form CO2 and H2O

Histamine Decarboxylation of histidine forms histamine, a reaction

catalyzed by histidine decarboxylase. Histamine has many physiological roles, including vasodilation and constriction of certain blood vessels. An overreaction of histamine can lead to bronchial asthma and other allergic reactions. In addition, histamine stimulates secretion of both pepsin and hydrochloric acid by the stomach, and is useful in the study of gastric activity

Serotonin 5-hydroxytryptamine (5-HT), also known as serotonin, results from

hydroxylation of tryptophan by a tetrahydrobiopterin-dependent enzyme, hydroxylase and decarboxylation by a pyridoxal phosphate-containing decarboxylase. 5-HT is a neurotransmitter in the brain and causes contraction of smooth muscle of arterioles and bronchiole

s.

polyaminesFigure 9-12

Polyamines are important in cell proliferation and tissue growth. They are growth factors for cultured mammalian cells and bacteria. Since polyamines bear multiple positive charges that can interact with polyanions such as DNA and RNA, and thus can stimulate synthesis of nucleic acid and protein.

chapter 7 catabolism of proteins. nutritional function of proteins functions: structural catalytic,...

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