Amino Acid Metabolism (Exam II)

Question 1

What are the essential amino acids?

Answer 1

Phe, Val, Thr, Trp, Ile, Met, His, Leu, Lys

PVT TIM HLL

Question 2

Strictly ketogenic amino acids

Answer 2

Leucine & Lysine

Question 3

Partly ketogenic amino acids

Answer 3

Phenylalanine
Isoleucine
Tryptophan
Tyrosine

Question 4

What enzyme and coenzyme are involved in transamination?

Answer 4

Transamination: the transfer of the α-amino group from an aa to α-ketoglutarate, producing an α-keto acid
and glutamate:

Freely reversible reaction catalyzed by ubiquitous enzymes called aminotransferases (transaminases); can be used to synthesize non-essential aa from α-keto acid precursors. Each aminotransferase requires the coenzyme pyridoxal phosphate. (PLP), a derivative of vitamin B6, covalently linked to a lys at the active site

Question 5

What enzyme and coenzyme are involved in oxidative deamination?

Answer 5

Oxidative deamination: removal of the amino group from glu, producing NH3 (ammonia) and α-KG. Coupled with
transamination, it provides a mechanism for removing amino groups as NH3 from most aa. Glu is the only aa that undergoes oxidative deamination to any appreciable extent.

Catalyzed by glutamate dehydrogenase (GDH), a mitochondrial enzyme. NAD or NADP is required as a coenzyme/cosubstrate. NAD+ is favored for the forward
catabolic reaction, NADPH for the reverse synthetic reaction.

The low E signal ADP is an allosteric activator of the enzyme GDH for the forward reaction, while GTP is an allosteric inhibitor. Reverse rx likely occurs only if [NH3] is high.

Question 6

Urea Cycle: Why? What is its function?

Answer 6

The UC of liver converts toxic ammonia (NH3) to non-toxic urea. The urea diffuses into plasma, and is excreted by the kidneys.

Question 7

Urea Cycle: Where? Where does it occur? Include the cellular and sub-cellular locations

Answer 7

The UC in the liver involves five enzymatic reactions, two mitochondrial and three cytoplasmic, and requires the hydrolysis of the equivalent of 4 ATP/1 urea produced.

Question 8

Urea Cycle: How? Be able to sketch it out using names, not structures.

Answer 8

1. Bicarbonate (from CO2) provides the carbon atom of urea. Free ammonia provides one of the nitrogen atoms of urea.
2. Rate-limiting formation of carbamoyl phosphate by carbamoyl phosphate synthetase I (CPSI). This enzyme requires N-acetylglutamate (NAG) from NAG synthase as an allosteric activator. The NH3 incorporated into carbamoyl phosphate is provided primarily by the oxidative deamination of glutamate by glutamate dehydrogenase, also a mitochondrial enzyme and abundant in liver cells.
3. Formation of citrulline from ornithine and carbamoyl phosphate, catalyzed by ornithine transcarbamoylase (OTC). Ornithine and citrulline are basic aa but not incorporated into cellular proteins. Why not? No codons. In this step an antiporter moves: ornithine for citrilline.
4. Citrulline condenses with asp to form argininosuccinate via argininosuccinate synthetase (ASS), driven by the hydrolysis of the third ATP to AMP and PPi. The asp is from transamination of glu by AST.
5. Formation of arginine and fumarate via the cleavage of argininosuccinate by a lyase (ASL). The production of fumarate links the Urea and TCA cycles
6. Formation of ornithine and urea via the cleavage of arginine by arginase-1, an enzyme virtually exclusive to the liver. Ornithine re-enters the cycle, while urea diffuses out into the plasma for excretion in urine.

Question 9

Urea Cycle: When? How is it regulated?

Answer 9

Regulation of the UC by:

a. changes in substrate concentration
b. activation of CPSI by NAG
c. changes in enzyme concentration

Question 10

For each molecule of urea made in the liver

Answer 10

a. Two N’s are eliminated, one from NH3 and one from asp. The asp N is from glu via transamination of OAA by AST.
b. Four high-energy bonds are hydrolyzed.
c. Ornithine is regenerated.

Question 11

Which UC enzyme is most likely deficient in a patient whose blood studies revealed: BUN of 2 mg/dL (normal, 8-25), ammonia of 887 µM (normal, 10-35), gln of 1611 µM (normal, 337-673), ala of 390 µM (normal, 136-440), citrulline of 1400 µM (normal, 10-34), argininosuccinate of 0 µM (normal, 0), and arg of 23 µM (normal, 30-124). Why might arg supplementation be of use in the treatment of the patient?

Answer 11

ASL deficiency as arginine and fumarate are formed via the cleavage of argininosuccinate by a lyase (ASL). Thus, citrulline is high (because it can’t go anywhere); glutamine is high (because of all the free ammonia), etc.

Question 12

Which UC enzyme is X-linked? Why might orotic acid be elevated if this enzyme is deficient?

Answer 12

Ornithine transcarbamoylase (OTC). The increased orotic acid concentrations result from the buildup of carbamylphosphate. This biochemical phenotype (increased ammonia, low citrulline and increased orotic acid) is classic for OTC deficiency.

Question 13

In what other nontoxic forms is NH3 transported?

Answer 13

Blood NH3 concentration is kept low, in part, because little is transported as free ammonia. Instead it is transported in non-toxic forms such as glutamine (gln).

Question 14

Where is BCAA catabolism begun? What is the enzyme involved? What coenzyme(s) is/are required?

Answer 14

The branched-chain, essential amino acids (leu, ile, val) from dietary sources are taken up preferentially by skeletal muscle, rather than by liver. Muscle responds to physiologic stress by degrading protein, with the release and catabolism of BCAA.

Transamination catalyzed by a single branched-chain amino acid aminotransferase, an enzyme plentiful in muscle but low in liver; α-KG → glu.

Coenzymes: TPP, CoA, lipoic acid, NAD+, FAD (all) Biotin, ATP (leucine), Biotin, B12 (valine & isoleucine)

Question 15

Treatment of OTC deficiency

Answer 15

Administration of compounds that bind non-essential aa for excretion. Examples include phenylacetate which binds gln, and benzoate which binds gly. Replacement (synthesis) of Gly and Gln uses NH3.

Question 16

What is the function of BCKAD? What coenzyme(s) does it require? If deficient, what pathology results?

Answer 16

The α-keto acids generated by transamination are oxidatively decarboxylated and attached to CoA by a single multi-enzyme complex, branched-chain α-keto acid dehydrogenase (BCKAD, also BCKD), present in most tissues but abundant in muscle. NAD+ is reduced to NADH.

1) The BCKAD complex is similar to PDH and α-KD in sub-cellular location, mechanism, and coenzyme requirements; all share a common E3. BCKAD, like PDH, is regulated by P/deP.
a) Active if deP.
b) FA β-oxidation and use of KB produce NADH and so likely inhibit BCKAD, sparing muscle protein.

A deficiency in BCKAD, leading to accumulation of branched-chain amino and keto acids with associated acidemia and aciduria, is known as Maple Syrup Urine Disease (MSUD) due to the characteristic odor of urine, sweat, tears, and feces.

In classic MSUD (

Question 17

Which BCAA is glucogenic, which is ketogenic, which is both?

Answer 17

Acetyl CoA (from leu and ile) that can be used in ketogenesis;

succinyl CoA (from val and ile) that can enter into the TCA cycle and serve as a substrate for gluconeogenesis.

Question 18

Which reducing equivalents are produced by the catabolism of BCAA? Where is each oxidized?

Answer 18

NAD+ → NADH (oxidative decarboxylation)
FAD → FADH2 (dehydrogenation)
NAD+ → NADH (formation of proponyl CoA)

Question 19

Outline the metabolism of propionyl CoA, being sure to include the required coenzymes. What are the inputs to
the propionyl CoA pool?

Answer 19

Valine & Isoleucine → Transamination (branched-chain amino transferase, αKG → glu) → Oxidative decarboxylation (branched chain alpha-ketoacid dehydogenase. Coenzymes: TPP, CoA, Lipoic acid, NAD+, FAD; NAD+ → NADH) → Dehydrogenation (FAD → FADH2) → Propionyl CoA → (with biotin & carboxylase) Methylmalonyl CoA → Succinyl CoA (B12 & mutase)

Question 20

What is the source of the ala and the gln produced by BCAA catabolism? Where and how is each used?

Answer 20

Ala is formed in muscle by transamination of the pyruvate produced from the succinyl CoA generated in the catabolism of val and ile. The ala is transported to the liver where glu and pyruvate reform. The pyruvate can be used by the liver as a substrate for gluconeogenesis. The glu can be deaminated; the NH3 is used in the UC. Ala, then, carries N to the liver

Gln is formed in muscle by the amidation of the glu produced, at least in part, in the transamination reaction for BCAA. Glutamine synthetase is up-regulated in response to glucocorticoids. The gln enters the blood and is metabolized by the gut, liver and kidney.

in the gut: gln is deamidated to glu + NH3 by glutaminase. The glu is converted to citrulline, which is sent out into the blood, picked up by kidney, and used for the synthesis of arg. The glu also can be transaminated with pyruvate by ALT, generating α-KG and ala, which are sent out into blood and used for gluconeogenesis in liver and kidney.

in the liver and kidney: also deamidated to glu + NH3 by glutaminase. The glu can be transaminated (as above) or oxidatively deaminated by mitochondrial GDH to NH3 + α-KG. The α-KG generated can be used in gluconeogenesis: α-KG to OAA via TCA cycle, OAA to PEP via PEPCK, PEP to glucose.

Question 21

What coenzyme/cosubstrate is made, in part, from trp? What is Hartnup Disorder? How does it differ from
pellagra? How are both treated?

Answer 21

In its catabolism, trp first undergoes an oxidative cleavage of the pyrrole ring, producing N-formylkynurine. Enzyme is heme-containing trp oxygenase. Hydrolytic removal of the formyl group yields kynurenine. Kynurenine can be catabolized to alanine and acetoacetyl CoA; primary pathway OR metabolized to quinolinate; quantitatively minor but physiologically
significant pathway. Quinolinate from trp, along with niacin (nicotinic acid, NA) supplied by diet, can be
converted to the coenzymes, NAD and NADP.

Hartnup disorder is a genetic defect (AR) in intestinal and renal absorption of trp. The clinical signs of Hartnup, when manifested, are similar to those of pellagra, a disease of dietary deficiency of NA (and trp), and include
photosensitive dermatitis, diarrhea, and intermittent ataxia. However, even homozygotes for Hartnup generally do not show signs (hence “disorder” rather than “disease”) since diet typically is adequate. When necessary, treated with oral niacin (as nicotinamide).

Question 22

What is the function of phenylalanine hydroxylase? What coenzyme does it require? If the enzyme is deficient,
what condition results? Why might patients with PKU have light complexion?

Answer 22

Phe (an essential aa) is irreversibly converted to tyr (non-essential aa) by hydroxylation at C4 through the action of the liver enzyme, phenylalanine hydroxylase (PAH). The reaction requires tetrahydrobiopterin (THB, BH4) as a coenzyme; gets oxidized to DHB (BH2). THB is synthesized from GTP.

A “complete” deficiency in phenylalanine hydroxylase leads to classical phenylketonuria (PKU), the most common disease of enzyme deficiency in amino acid metabolism. As a consequence of this enzyme deficiency, normally minor pathways become major ones, with products such as phenylpyruvate (a phenyl α-keto
acid) appearing in the urine, hence “PKU.” Phenylacetate give the urine a characteristic “mousey” odor. Tyrosine is a precursor for catecholamines (dopamine, epinephrine, and norepinephrine), via tyrosine hydroxylase + THB, and for melanin via tyrosinase. T3, T4 synthesis from tyr also.

Question 23

Outline metabolism of acetoacetate & acetyl CoA from BCAA cycle.

Answer 23

Leucine → Transamination (branched-chain amino transferase, αKG → glu) → Oxidative decarboxylation (branched chain alpha-ketoacid dehydogenase. Coenzymes: TPP, CoA, Lipoic acid, NAD+, FAD; NAD+ → NADH) → Dehydrogenation (FAD → FADH2) → 3-Methylcrotonyl CoA → (with ATP, biotin & carboxylase) 3-Methylglutaconyl CoA → HMG CoA → Acetoacetate & Acetyl CoA

Question 24

What is alcaptonuria? What enzyme is deficient? What are the signs of alcaptonuria?

Answer 24

Alcaptonuria, an AR condition caused by a deficiency in homogentisate oxidase which is involved in tyrosine degradation by cleaving the ring.

The homogentisate that accumulates oxidizes to form
a dark pigment-like polymer that is seen in urine and is deposited in bone and connective tissue (ochronosis).

This pigment is thought to be responsible for
the joint destruction and deterioration of cardiac valves seen at an early age.

Question 25

What two amino acids are required for the synthesis of cys? Which one supplies the S?

Answer 25

Cysteine is made from met and ser. The sulfur is donated by methionine.

Question 26

In what structural way are met and hcy different?

Answer 26

Methionine has a methyl group that homocysteine is lacking. The source of the -CH3 is primarily N5
-methyltetrahydrofolate (THF). A less common source is betaine (trimethylglycine).

Question 27

What is the function of cystathionine synthase? What is a consequence of its deficiency?

Answer 27

Hcy is condensed with ser to form cystathionine catalyzed by cystathionine synthase. The cystathionine is hydrolyzed by cystathionase to cysteine and α- ketobutyrate plus NH3. Both reactions require PLP.

The α-ketobutyrate formed is oxidatively decarboxylated to propionyl CoA which in turn gives rise to succinyl CoA

Elevated levels (> 16 µmol/L) of homocysteine in the blood have been associated with increased risk for premature occlusive vascular disease such as stroke, MI, and peripheral vascular disease. Elevated hcy is thrombogenic: damages endothelium, activates
the clotting cascade and inhibits fibrinolysis; see platelet
activation and smooth muscle proliferation.

Question 28

Inputs to propionyl CoA pool

Answer 28

VOMIT: val, odd-numbered fa, met, ile, thr

Question 29

What two reactions in the body are known to require vitamin B12? Which of the two also requires THF?

Answer 29

MTR, also known as methionine synthase, is a methyltransferase enzyme, which uses the B12 and reaction type 2 to transfer a methyl group from 5-methyltetrahydrofolate to homocysteine, thereby generating tetrahydrofolate (THF) and methionine. This requires N5-methyltetrahydrofolate (THF).

MUT is an isomerase which uses the B12 to catalyze a carbon skeleton rearrangement (the X group is -COSCoA). MUT’s reaction converts MethylMalonyl-CoA to Succinyl-CoA, an important step in the extraction of energy from proteins and fats.

Question 30

What is meant by the “folate trap?”

Answer 30

Hcy can be remethylated to met: The source of the -CH3 is primarily N5-methyltetrahydrofolate (THF). A less common source is betaine (trimethylglycine). It is an unique reaction in that it is the only reaction in which THF carries and donates a methyl group; B12 is recipient, becomes methylcobalamin that donates methyl to hcy. If B12 is deficient, THF is trapped in the nonutilizable N5-methyl form: folate trap. Hcy concentration increases.

Question 31

How might deficiencies in B6, B12 and folate lead to an increase in [hcy]?

Answer 31

B6: Hcy is condensed with ser to form cystathionine catalyzed by cystathionine synthase. The cystathionine is hydrolyzed by cystathionase to cysteine and α- ketobutyrate plus NH3. Both reactions require PLP which is a derivative of B6.

B12 & folate: If B12 is deficient, THF is trapped in the nonutilizable N5-methyl form: folate trap. Hcy concentration increases.

Question 32

Source & Function: SAM

Answer 32

Source: Met condenses with ATP, forming S-adenosylmethionine (SAM or AdoMet).

Function: -CH3 group in SAM is activated and is able
to be transferred to a variety of acceptors via methyl transferases. Acceptors include phospholipids, proteins, nucleic acids, biogenic amines, etc.

Question 33

Source & Function: THF

Answer 33

Source: THF is formed from folic acid in a two-step, NADPH-requiring reduction reaction catalyzed by an enzyme of the intestinal mucosa, dihydrofolate reductase. The THF gets methylated at N5, forming N5-methyltetrahydrofolate, the major form in blood (bound to albumin).

Function: Converts hcy to met

Question 34

Source & Function: biotin

Answer 34

Biotin, a water-soluble B-complex vitamin, is the prosthetic group of most enzymes involved in carboxylation reactions. These include acetyl CoA
carboxylase, propionyl CoA carboxylase, pyruvate carboxylase, and methylcrotonyl CoA carboxylase

Degradation of holoenzymes produces biocytin (biotin-lys) that is acted upon by biotinidase, which cleaves bond between biotin and the lys residue; biotin gets reutilized.

Question 35

Source & Function: PAPS

Answer 35

Cysteine can be degraded to pyruvate and sulfate. The
sulfate can be used to make 3′-phosphoadenosine-5′-phosphosulfate (PAPS), the source of sulfate groups for
addition to biological molecules such as GAGS.

Question 36

Source & Function: PLP

Answer 36

Coenzyme pyridoxal phosphate (PLP), a derivative of vitamin B6. PLP acts as a coenzyme in all transamination reactions, and in certain decarboxylation, deamination, and racemization reactions of amino acids.

Question 37

Source & Function: THB

Answer 37

THB is synthesized from GTP. A naturally occurring essential cofactor of the three aromatic amino acid hydroxylase enzymes, used in the degradation of amino acid phenylalanine and in the biosynthesis of the neurotransmitters serotonin (5-hydroxytryptamine, 5-HT), melatonin, dopamine, norepinephrine (noradrenaline), epinephrine (adrenaline), and is a cofactor for the production of nitric oxide (NO) by the nitric oxide synthases

Question 38

N5-CH3 THF is the major form of THF in the blood. The methyl group is used in the B12-dependent conversion
of hcy to met. What is the fate of the THF?

Answer 38

THF becomes demethylated. It changes from N5-methyltetrahydrofolate to tetrahydrofolate

Question 39

What is the role of N5, N10-methylene THF? Of N10-formyl THF?

Answer 39

N5, N10-methylene THF: helps thymidylate synthetase convert deoxyuridine monophosphate (dUMP) to deoxythymidine monophosphate (dTMP).

N10-formyl THF: synthesis of the purine bases

Question 40

What is meant by “multiple carboxylase deficiency”?

Answer 40

Defects in holocarboxylase synthetase (early-onset form: birth to 1 month) and in biotinidase (late-onset form: several months of age) result in multiple carboxylase deficiency. Clinical signs are neurologic and cutaneous; treatment is oral biotin.

Apocarboxylase is biotinated by holocarboxylase synthetase. Without holocarboxylase synthetase, a lot of holocarboxylases do not work:
propionyl CoA carboxylase (amino acids, fatty acids)
acetyl CoA carboxylase (fatty acid synthesis)
pyruvate decarboxylase (gluconeogensis)
methylcrotonyl CoA carboxylation (leu degradation)

Question 41

What is the rationale for newborn screening?

Answer 41

NBS allows for early detection and treatment (key) of potentially fatal or disabling conditions