Amino acid metabolism I

proteinsproteins

amino acidspool

proteosynthesis specificspecificsynthesissynthesis((nonessencialnonessencialAA)AA)

keto acids NH3

CO2, H2O, ATP physiologicallyphysiologicallyimportantimportantnitrogenousnitrogenouscompoundscompounds

specific pathways↓

hormonesneurotransmiters

koenzymesporphyrins

purinespyrimidines

creatine

biogenic amines

proteolysis(digestion)

common metabolic pathways

TCAUC

urea

glukose ketone bodies

H2N-C-NH2

O

transamination deamination decarboxylation

↓Phe ↓↓ Tyr ↓

Lys ↓, Arg ↓Phe ↓, Tyr ↓, Trp ↓, Met ↓, Leu ↓Ala ↓, Gly ↓,Ser ↓A: ↓, Ala ↓ Ile, ↓ Leu, ↓ValB: ↓ Lys, ↓ Arg

endopeptidasses

exopeptidases

Protein digestion - proteolytic enzymes (proteinases/proteases)

stomach

smallintestine

brush bordermembrane enzymes

absorption - enterocyte (active transport)portal blood liver

TrypsinChymotrypsinElastase

Carboxypeptidases

Specificity and activation of pancreatic proteases

Trypsin = activator of all digestive proteolytic enzymes in the small intestine

Proteinases (proteases)

1. Synthesis and secretion: proenzymes (zymogenes)

2. Activation: cleavage of several peptide bonds or/and removal ofa short peptide→ openning of an active site

pepsinogen pepsin + 41 AA

trypsinogen trypsin + 6 AA

3. Inhibition: protein inhibitors→ interaction with proteinases active sites

HCl

enteropeptidase

trypsin

trypsinogen

substrate(polypeptide)

• pancreatic trypsin-inhibitor:small protein → very tight binding at the activesite of trypsinu ⇒ inactive complex (half life: several months)

↓protection of intestinal walls and cellsagainst proteolytic cleavage

• α1-antitrypsin (α1-antiproteinase, antielestase):plasma protein – irreversible binding at the active site of trypsin and elastase→ protection of tissues against digestion by elastase (trypsin)

genetic mutants↑

smokerssmokers

low blood level↓

destruction of alveolar walls(degradation of connective structures by elastase)

↓emphysema

Proteinases inhibitors

slow secretion from liver

Protein turnover

Dietary proteins 100 g/day equivalent amount of nitrogenousmetabolites (mainly urea) excreted

Body proteins 400 g/day constant degradation and resynthesis

AA – mostly metabolised in liver; Leu, Ile, Val pass without transformation –utilized in muscle and brain

glutamine, valine, alanine, glycine- most abundant AA in the blood circulation

biological half-life of proteins:

< 2 h digestive enzymes, ornithine dekarboxylase, HMG-CoA-reductase< 2- 200 h most proteins> 200 h hemoglobin, acetylcholine receptorseveral months, collagen, structural proteins

years

Intracellular protein degradation

1. Lysosomal proteases (cathepsins, collagenase,dipeptidases..):most extracellular proteins, long-lived

proteins2. Proteasomes:abnormal proteins, short-lived proteins

Ubiquitinationcytosolic protein ubiquitin –kisskiss ofof deathdeath- binding with a protein predestines it for degradation inproteasome= oligomer – supramolecular structure, severalsubunits = proteinases

ubiquitin-COOH + H2N-Lys-protein

mark for degradation

Signals for degradation:1.oxidation: Lys, Trp, His, Cys2. PEST-sequence: Pro, Glu, Ser, Thr3. NH3-end of AA: Arg, Lys, Asp, Phe

Catabolism of amino acids – common reactions

Pyridoxal phosphate– unique role in amino acid metabolism - cofactor ofenzymes catalyzing different kinds of AA transformations

in the active site held by noncovalent interactions orlinked covalently to ε-amino group of lysine

pyridoxal phosphate (PLP)pyridoxine Schiff baseAA - PLP

3

21

essential intermediate of AA metabolism

a derivative of vitamin B6

1. Decarboxylation2. Transamination3. Transformation of a carbon chain→ specific for each amino acid

common reactions of amino acid metabolism

1. Decarboxylation biogenic amines; enzymes - decarboxylases

R-C-COOH R-CH2-NH2 + CO2

neuromediators

H

NH2

histidine → histamine: mediatorserine→ ethanolamine→ phospholipidscysteine → cysteamine→ CoA-SHTrp → 5-OH-Trp → serotonineglutamic acid→ GABA

PLP

2

putrescine

Polyamines– spermidine, spermine– initial substrates ornithine and methioninestabilization of RNA, DNA, membranes - regulation of cell growthand proliferation, regeneration of tissues

ornithinedecarboxylase

2. Transamination→ keto acids; enzymes – aminotransferases! reaction reversible – involved both in catabolism andbiosynthesis of aminoacids

H

NH2

R2-C-COOH

O

R2-C-COOH

H

NH2

R1-C-COOH PLP+ R1-C-COOH

O

+

Example: alanine aminotransferase

alanine 2-oxoglutarate pyruvate glutamate= α-ketoglutarate

-

--

-

exclusive acceptor of amino group in transaminase -NH2 of any AA incorporatedreaction in AA catabolism AK into molecule of glutamate

1 1

1

2

1

2

2

2

Mechanism oftransamination

ALT – alanine aminotransferase:

alanine + 2-oxoglutarate pyruvate + glutamate

AST – aspartate aminotransferase:

aspartate+ 2-oxoglutarate oxaloacetate+ glutamate

Localization: mainly liver, muscle, kidneyALT – cytosol, AST –cytosol and mitochondria!

Metabolic significance:ALT, AST – catabolism andi biosynthesis of alanine/aspartate,ALT – important for utilization of muscle protein AA for gluconeogenesis

(glucose-alanine cycle)AST – replenishment of oxalacetate for CC (anaplerotic reaction),

transport of oxalacetate across mitochondrial membrane (gluconeogenesis),important for the function of malate dehydrogenase shuttle and urea syntesis(formation of aspartate as a donor of one nitrogen atom ofthe urea)

Diagnostic value:ALT, AST – markers of liver injury (hepatocellular necrosis),AST – blood level increases at myocardial infarction (not used as a marker in the present

IM diagnostics)

ALT, AST – important aminotransferases/transaminases

PLP

PLP

Deamination: removal of -NH2 as NH3

1. Direct deamination→ serine, threonine serine (threonine) dehydratase

2. Oxidative deamination– other amino acids

L-amino acid oxidases, cofactor FMN

- liver, kidney, low activity – little valuefor AA metabolism

D-amino acid oxidases, cofactor FAD

- ?deamination of D-amino acidsof bacterial and plant origin

CH2-C-COOH

NH2OH

H

CH2= C-COOH

NH2

NH3CH3-C-COOH

O

+ CH2=C-COOH

OH

H2O H2O

OHH

Oxidative deamination of glutamate - kee reaction for removal of nitrogenfrom amino acids

amino acid+α-ketoglutarate→ keto acid + glutamate -a product of transamination

NH3 + α-ketoglutarate

! Glutamate dehydrogenase (GDH) (NAD+/ NADP+)(high amount – liver mitochondria – used in diagnostics of liver

diseases)

HOOC-CH2-CH2-C-COOH

HOOC-CH2-CH2-C-COOH HOOC-CH2-CH2- C-COOH + NH3

NAD+

NADH+H+

H2ONH2

NHO

H2

O

α−ketoglutarate

glutamate

oxidative deamination

Ammonia - NH3• sources:

1. Deamination of amino acids (glutamate) –all tissues2. Hydrolysis of glutamine -kidney, small intestine, liver3. Bacterial degradation of proteins and urea (urease) in theintestine4. Catabolism of purines, pyrimidines, catecholamines- liver, brain

• detoxification:1. Urea synthesis -!liver! (exclusively) → kidney→ urine2. Synthesis ofglutamine – extrahepatic tissues3.Formation ofNH4

+ - kidney → urine

HOOC-CH-CH2-CH2-COOH

NH2 NH2

O

NH2

HOOC-CH-CH2-CH2-CNH3 +glutamate glutamine

glutaminesynthetase

glutamineglutaminase

NH3 + glutamate

muscle, brain

kidney

H+NH4

+

ad 2, 3

urine

kidney, liver

Ammonia metabolism - overview

Urea synthesis = Ureagenesis:major pathway of NH3 detoxification

Localization: liver⇒ mitochondria, cytosol of periportal hepatocytes

precursors: NH3oxidative deamination

of glutamate (GDH)CO2

TCA cycle

aspartatetransamination ofoxaloacetate (AST)

glutamate

glutamateNH3 + α-ketoglutarate

aspartateα-ketoglutarate

oxaloacetate

NAD+

Energy needs of ureagenesis: 4 ATP

H2N-C-NH2

O

NADH+H+

GDH

AST

Proteins↓↓↓↓↓↓↓↓

amino acidsaminotransferase

α-keto acid

glutamate

α-ketoglutarate

glutamate degydrogenase

NAD+

NH3 aspartate

NADH+H+

α-ketoglutarate

oxaloacetate

α-ketoglutarate

aspartate aminotransferase

citrulline

argininosuccinate

fumaratearginineurea

ornithine

carbamoyl phosphate

ureaureacyclecycle

CO2

Urea cycle – formation of urea

- NH3 enters intu the cycle after formationof carbamoyl phosphate

- carbamoyl phosphate formation fromNH3, CO2, ATP catalyzescarbamoylphosphate synthetase I

– enzyme located in hepatocytemitochondriaonly !

-carbamoyl phosphateis also precursorof pyrimidine nucleotides – formation incytosol – catalyzed by carbamoylphosphate synthetase II,source of its NH2-group is glutamine(! not NH3)

Cooperation of urea cycle and citric acid cycle- utilization of fumarate, a side product of ureagenesis

oxaloacetate

Regulation of urea synthesis

1. A substrate delivery- low-protein diet - decreased AA intake decreased NH3 formation

low activity of urea cycle, decreased urea level in urine

- high-protein diet, fasting (degradation of muscle proteins) - increased AA deliveryinto liver increased activity of urea cycle

2. N-acetylglutamate– allosteric activator of carbamoyl phosphate synthetase I (essential for the enzyme activity)formation from glutamate – increased at higher AA delivery= at protein degradation -explanation of increased

ureagenesis in fasting

Genetic defects of urea cycle enzymeshyperammonemia – partial deficit can be treated by low-protein diet and

suplementation of food with arginine (precursor of ornithine)- significantly decreased activities of ureagenetic enzymes -incompatible with life

Hyperammonemia

1. liver cirrhosis (alcoholic), hepatitis, obstruction ofbile duct⇒ NH3 from GIT enters via portal blood→ directly into systemic circulation

2. genetic defects of enzymes of ureagenesis→ inhibition of urea synthesis→ NH3 cannot be detoxified⇒ vomiting, lethargy, coma , serious brain

damage, death

Ammonia toxicity (namely forCNS) –suggested mechanisms

Brain: NH3 + α−ketoglutarate glutamate

decreased mitochondrial level→ inhibition of citric acid cycle(substrate depletion)

→ decreased ATP synthesisNH3 + glutamate glutamine

depletion of glutamate (neurotransmitter)

(excess of NH3 inhibits glutaminase)

alteration of nerve impuls transmission

GDH

NADPH+H+ NADP+

x

Fate of ammonia in tissues

brain + most tissues(also muscle)

amino acid

transamination

glutamate

GDHNH3

glutamine synthetase

glutamine

Liver

amino acid

transamination

glutamate

GDHNH3

urea cycle

urea

Kidneyglutamine

glutaminase

glutamate + NH3 urine

urea

Glucose-alanine cycle

- connection of muscle glycolysis andliver gluconeogenesis – analogy to Cori cycle

- kee role ofALT

- important in starvation- gluconeogenesis from AA - dominant- uses also alanine released during breakdownof contractile proteins