Chapter 18

Amino Acid Oxidation and The Production of Urea

Amino Acid Oxidation

Dependency of amino acid as energy source

Carnivores> herbivores> microorganism >> plant

Amino acid degradation in animals Amino acids for oxidation

Extra amino acid during protein turnover Protein-rich diet (no storage) During starvation or in uncontrolled diabetes

Removal of amino group (NH4+)

-keto acid (C skeleton of amino acids)

Oxidation to CO2 & H2O

Sources of C3 or C4 units for gluconeogenesis or fuels

Pathways of amino acid catabolism

18.1 Metabolic Fates of Amino Groups

Amino Group Catabolism

Amino Group Catabolism

Amino acid metabolism amino group (= nitrogen metabolism)

Liver is a major site Recycle for biosynthetic pathways Excretion; ammnonia, urea, uric acid

Glutamate & glutamine General collection point for amino group NH3 from amino acids + -ketoglutarate glutamate

into mitochondria, release of NH4+

Source of ammonia Dietary protein (major source) Muscle & other tissues

NH4+ + glutamate glutamine

mitochondria in hepatocytes

NH4+ + pyruvate alanine hepatocytes

Digestion of Dietary Protein

In stomach Entry of diet secretion of gastrin from gastric mucosa secretion of HCl (parietal cells), pepsinogen (chief cells)

Acidic gastric juice (pH 1.0 to 2.5) Antiseptic & denaturing agent (protein unfolding)

Pepsinogen : zymogen Conversion to active pepsin by autocatalytic cleavage (at low pH) Digestion of peptide bonds at Phe, Trp, Tyr mixture of small peptides

In small intestine Low pH secretion of secretin stimulation of bicarbonate secretion from pancreas neutralization Arrival in the upper part of intestine (duodenum) release of cholecystokinin into blood

stimulation of pancreatic zymogens Trypsinogen : activated by enteropeptidase Chymotrypsinogen, procarboxypeptidase A and B : activated by trypsin

c.f.) Protection of pancreas from proteolytic digestion Production of zymogens Pancreatic trypsin inhibitor

Protein digestion by trypsin, chymotrypsin, carboxypeptidase, aminopeptidase Uptake of amino acids by the epithelial cells

Digestion of Dietary Protein

Blood capillariesLiver

Transamination

1st step of amino acid catabolism Transfer of -amino group to -ketoglutarate

Generation of L-glutamate & -ketoacid

Aminotransferase (transaminase)

Amino acid specificity (named after amino acids)

Reversible reaction

; ∆G’° ≈ 0 kJ/mol

Pyridoxal phosphate (PLP)

Bimolecular Ping-Pong reactions

Pyridoxal phosphate (PLP)

Coenzyme form of pyridoxine (vitamin B6)

Intermediate carrier of amino group

Electron sink for carbanion (resonance stabilization) Transamination Racemization (L- & D-form interconversion) Decarboxylation

PLP-mediated transamination at -carbon

PLP-mediated transamination: Ping-Pong mechanism

amino acid -ketoglutarate

pyridoxal phosphate pyridoxamine phosphate pyridoxal phosphate

-keto acid glutamate

PLP-mediated amino acid transformations at -carbon

Oxidative Deamination of Glutamate

Oxidative deamination Mitochondrial matrix of hepatocytes Glutamate dehydrogenase

Generation of -ketoglutarate & ammonia NAD+ or NADP+ as electron acceptor Allosteric regulation

By ADP (inhibition) By GTP (activation)

Transdeamination Transamination of A.a. + oxidative deamination of Glu A few amino acids undergoes direct oxidative deamination

Glutamine as Ammonia Carrier in the Bloodstream

Ammonia generated in extrahepatic tissues

Glutamine synthetase Incorporation of ammonia into

glutamate glutamine

Transport of gln to the liver via blood

Higher gln concentration than other amino acids in blood

Glutaminase in the liver, intestine, and kidney

Glutamine Glutamate + NH4+

Alanine Transports Ammonia from Skeletal Muscles to the Liver

Glucose-alanine cycle In muscle

Glycolysis & degradation of amino acids Alanine aminotransferase

Transfer amino group of glutamate to pyruvate alanine + -ketoglutarate

Transport of alanine to the liver

In the liver Alanine aminotransferase

Transfer amino group of alanine to -ketoglutarate glutamate + pyruvate

Gluconeogenesis Pyruvate , lactate glucose

Transport of glucose to muscle

Ammonia is toxic to animals.

Comatose state of brain (high brain’s water content)

1. NH3: alkalization of cellular fluid 2. -ketoglutarate, NADH, ATP:

citric acid cycle & ATP production3. glutamate and GABA (-aminobutyrate):

neurotransmitter depletion

18.2 Nitrogen Excretion and the Urea Cycle

Produced in liver

Blood

Kidney urine

Urea Cycle in Mitochondria

Formation of carbamoyl phosphate; preparatory step

NH4+ + HCO3

- + 2 ATP carbamoyl phosphate + 2 ADP + Pi

Carbamoyl phosphate synthetase I

- ATP-dependent reaction

1st step in the urea cycle;

Ornitine + carbamoyl phosphate citrulline + Pi

Ornitine transcarbamoylase

Urea Cycle in Cytosol

2nd step; formation of argininosuccinate Incorporation of the second N from aspartate Argininosuccinate synthetase ATP requirement Citrullyl-AMP intermediate

3rd step; formation of arginine & fumarate Argininosuccinase; only reversible step in the cycle

4th step; Cleavage of arginine to urea & ornithine Arginase

Asparatate-argininosuccinate shunt

Metabolic links between citric acid and urea cycles In cytosol

Fumarate to malate citric acid cycle in mitochondria In mitochondria

OAA + Glu -ketoglutarate + Asp urea cycle in cytosol

Energetic cost

• Consumption

3 ATP for urea cycle

• Generation

Malate to OAA

1 NADH = 2.5 ATP

Regulation of the Urea Cycle

Long term regulation

Regulation in gene expression

Starving animals & very-high protein diet Increase in synthesis of enzymes

in urea cycle

Short term regulation

Allosteric regulation of a key enzyme

Carbamoyl phosphate synthetase I Activation by N-acetylglutamate

Treatment of genetic defects in the urea cycle

Genetic defect in the urea cycle

ammonia accumulation; hyperammonemia Limiting protein-rich diet is not an option Administration of aromatic acids; benzoate or phenylbutyrate Administration of carbamoyl glutamate Supplement of arginine

18.3 Pathways of Amino Acid Degradation

Amino Acid Catabolism

Carbon skeleton of 20 amino acids

Conversion to 6 major products

- pyruvate

- acetyl-CoA

- -ketoglutarate

- succinyl-CoA

- fumarate

- oxaloacetate

Glucogenic or Ketogenic Amino Acids

Ketogenic amino acids

Conversion to acetyl-CoA or acetoacetyl-CoA

ketone bodies in liver

Phe, Tyr, Ile, Leu, Trp, Thr, Lys Leu : common in protein

Contribution to ketosis under starvation conditions

Glucogenic amino acids

Conversion to pyruvate, -ketoglutarate, succinyl-CoA, fumarate, and OAA

glucose/glycogen synthesis

Both ketogenic and glucogenic Phe, Tyr, Ile, Trp, Thr

Enzyme cofactors in amino acid catabolism

One-carbon transfer reactions ; common reaction type, involvement of one of 3 cofactors

Biotin ; one-carbon tranfer of most oxidized state, CO2

Tetrahydrofolate (H4 folate) ; One-carbon transfer of intermediate oxidation states or methyl groups S-adenosylmethionine ; one-carbon transfer of most reduced state, -CH3

Tetrahydrofolate

folate (vitamin) to H4 folate Dihydrofolate reductase

Primary source of one-carbon unit

Carbon removed in the conversion of Ser to Gly

Oxidation states of H4 folate ; One-carbon groups bonded to

N-5 or N-10 or both- Methyl group (most reduced)- Methylene group- Methenyl, formyl, formimino group

(most oxidized) Interconvertible & donors of one-

carbon units (except N5-methyl-tetrahydrofolate)

S-adenosylmethionine (adoMet)

Cofactor for methyl group transfer Synthesized from Met and ATP

Methionine adenosyl transferase Unusual displacement of triphosphate from ATP

Potent alkylating agent Destabilizing sulfonium ion inducing nucleophilic attack on methyl group

Six amino acids are degraded to pyruvate

Ala, Trp, Cys, Ser, Gly, Thr pyruvate acetyl-CoA citric acid cycle or gluconeogenesis

Interplay of PLP and H4folate in Ser/Gly metabolism

3rd pathway of glycine degradation - D-amino acid oxidase detoxification of D-amino acid high level in kidney

- Oxalate crystals of calcium oxalate (kidney stones)

Seven Amino Acids Are Degraded to Acetyl-CoA

Trp, Lys, Phe, Tyr, Leu, Ileu, Thr acetoacetyl-CoA acetyl-CoA

Intermediates of Trp catabolism be precusors for other biomolecules

Catabolic pathways for Phe & Tyr

Phe & Tyr are precusors dopamine norephinephrine, epinephrine melanin

Phenylalanine hydroxylase

Mixed function oxidase ; Substrate hydroxylation + oxygen reduction to H2O Tetrahydrobiopterin as a cofactor

Example of A.a. metabolism defects; Phe catabolism

Phe degradaion fumarate + acetoacetyl-CoA Defects in Phe catabolism

Phenylketonuria (PKU) Genetic defect in Phe hydroxylase or

dihydrobiopterin reductase Elevated levels of Phe & phenylpyruvate Mental retardation

Alkaptonuria Genetic defect in homogentisate dioxygenase Oxidation of accumulated homogentisate

Black urine Arthritis

Genetic defects of A.a. metabolism defective neural development & metal retardation

Five Amino Acids Are Converted to -ketoglutarate

Pro, Glu, Gln, Arg, His; amino acids with five C skeleton converging to Glu

Four Amino Acids Are Converted to Succinyl-CoA

Met, Ileu, Thr, Val converging to propionyl-CoA

Branched-Chain Amino Acids Are Not Degraded in the Liver

Leu, Ile, Val

Primarily oxidized as fuels in muscle, adipose, kidney, brain

Branched-chain aminotransferases (not in liver)

Branched-chain -keto acid dehydrogenase complex

Asn and Asp are Degraded to Oxaloacetate