Amino Acid Catabolism

Molecular Biochemistry II

Amino Acid Catabolism: Nitrogen

Contents of this page:
Transaminase (amino transferase)
Deamination of amino acids
Formation of carbamoyl phosphate
Urea Cycle
Other roles of Urea Cycle intermediates

Transaminases (aminotransferases) catalyze the reversible reaction shown at right. There are multiple transaminase enzymes which vary in substrate specificity. Some show preference for particular amino acids or classes of amino acids as amino group donors and/or for particular a-keto acid acceptors.

An example of a transaminase reaction is shown at right.

Aspartate donates its amino group, becoming the a-keto acid oxaloacetate.
a-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 a-ketoglutarate.

Transaminases equilibrate amino groups among available a-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.

Although the amino N of one amino acid can be used to synthesize another amino acid, nitrogen must be obtained in the diet as amino acids (proteins).

Essential amino acids must be consumed in the diet because mammalian cells lack the enzymes to synthesize their carbon skeletons (a-keto acids). These include:

Isoleucine, leucine, & valine

Lysine

Threonine

Tryptophan

Phenylalanine (Tyrosine can be made from phenylalanine.)

Methionine (Cysteine can be made from methionine.)

Histidine (Essential for infants.)

The prosthetic group of the transaminase enzyme is pyridoxal phosphate (PLP), a derivative of vitamin B₆. (p. 986).

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

The a-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 a-keto acid. The amino group remains on what is now pyridoxamine Phosphate (PMP).

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

For more details about the reaction mechanism, and the postulated role of molecular strain in catalysis, see the article by Hayashi et al.

Several other enzymes that catalyze metabolism or synthesis of amino acids also utilize PLP as prosthetic group, and have mechanisms involving a Schiff base linkage of the amino group to PLP.

Chime Exercise: Two neighboring students or student groups should team up, each displaying, as recommended, one of the following:

Transaminase with PLP in Schiff base linkage to the active site lysine residue.
Transaminase in the PMP form, with glutarate, an analog of a-ketoglutarate, at the active site.

Students should then show and explain the structure displayed by them to the neighboring student or student group.

Transaminase-PLP

Transaminase-PMP

In addition to equilibrating amino groups among available a-keto acids, transaminases funnel amino groups from excess dietary amino acids to those amino acids (e.g., glutamate) that can be deaminated. Carbon skeletons of deaminated amino acids can be catabolized for energy or used to synthesize glucose or fatty acids for energy storage.

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 a-carbon is followed by hydrolysis, releasing NH₄⁺. (See diagram at right and on p. 989.)

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

Some other pathways for deamination of amino acids:

1. Serine Dehydratase catalyzes:
serine à pyruvate + NH₄⁺

2. L- & D-amino acid oxidases within peroxisomes catalyze:

Amino acid + FAD + H₂O à a-keto acid + NH₃⁺ + FADH₂
FADH₂ + O₂ à FAD + H₂O₂
Catalase then catalyzes: 2 H₂O₂ à 2 H₂O + O₂

Most terrestrial land animals convert excess nitrogen to urea prior to excreting it. Urea is less toxic than ammonia.

Urea Cycle occurs mainly in liver. The 2 nitrogen atoms of urea enter the Urea Cycle as NH₃ (produced mainly via the Glutamate Dehydrogenase reaction) and as the amino N of aspartate.

NH₃ (amino N) and HCO₃^- (carbonyl C) that will be part of urea are incorporated first into carbamoyl phosphate.

Carbamoyl Phosphate Synthase (Type I) catalyzes a three-step reaction with carbonyl phosphate and carbamate intermediates, as shown at right.

The reaction, which involves cleavage of 2 ~P bonds of ATP, is essentially irreversible.
Ammonia is the nitrogen input for this reaction of the Urea Cycle.

Alternate forms of Carbamoyl Phosphate Synthase, designated Types II and III, initially generate ammonia by hydrolysis of glutamine. The type II enzyme includes a long internal tunnel through which ammonia and reaction intermediates such as carbamate pass from one active site to another.

Carbamoyl Phosphate Synthase is the committed step of the Urea Cycle, and is subject to regulation.

Carbamoyl Phosphate Synthase has an absolute requirement for an allosteric activator N-acetylglutamate (p. 995). This derivative of glutamate is synthesized from acetyl-CoA and glutamate when cellular [glutamate] is high, signaling an excess of free amino acids due to protein breakdown or dietary intake.

The Urea Cycle is summarized in the diagram at right and on p. 992.

In addition to Carbamoyl Phosphate Synthase, one Urea Cycle enzyme is localized in the mitochondria:

1. Ornithine Transcarbamylase.

The 3 remaining Urea Cycle enzymes are in the cytosol:

2. Argininosuccinate Synthase
3. Argininosuccinase
4. Arginase

Given the localization of enzymes, for each cycle citrulline must leave the mitochondria, and ornithine must enter the mitochondrial matrix.

An ornithine/citrulline transporter in the inner mitochondrial membrane facilitates the necessary transmembrane fluxes or citrulline and ornithine.

A complete Krebs Cycle functions only within mitochondria. However, cytosolic isozymes of some Krebs Cycle enzymes are involved in regenerating aspartate from fumarate, as shown on p. 992.

Fumarate (produced by cleavage of argininosuccinate) is converted to oxaloacetate via Krebs Cycle enzymes Fumarase and Malate Dehydrogenase.
Oxaloacetate is converted to aspartate via transamination, e.g., from glutamate (see above). Aspartate then reenters the Urea Cycle, carrying an amino group derived from another amino acid.

Hereditary deficiency of any of the Urea Cycle enzymes leads to hyperammonemia - elevated [ammonia] in blood. Total lack of any Urea Cycle enzyme is lethal. Elevated ammonia is very toxic, especially to the brain. If not treated immediately after birth, severe mental retardation results.

Postulated mechanisms for toxicity of high [ammonia]:

High [ammonia] would drive the Glutamine Synthase reaction (p. 1033).
glutamate + ATP + NH₃ à glutamine + ADP + P_iThis would deplete glutamate, a neurotransmitter and the precursor for synthesis of GABA, another neurotransmitter.
Depletion of glutamate, as well as the high ammonia level, would drive the Glutamate Dehydrogenase reaction to reverse.
glutamate + NAD(P)⁺ ß a-ketoglutarate + NAD(P)H + NH₄⁺
The resulting depletion of a-ketoglutarate, an essential Krebs Cycle intermediate, could impair energy metabolism in the brain.

Treatment of deficiency of Urea Cycle enzymes (some treatments depend on which enzyme is deficient):

limiting protein intake to the amount barely adequate to supply amino acids for growth, while adding to the diet the a-keto acid analogs of essential amino acids.
Liver transplantation has also been used, since liver is the organ that carries out Urea Cycle.

Explore information about such genetic diseases in the OMIM web site (Online Mendelian Inheritance in Man). Links are provide here to information relating to hereditary deficiencies of:

Carbamoyl Phosphate Synthase and Ornithine Transcarbamylase.

Use the menu at the top of the OMIM page to change the display to Clinical Synopsis or Detailed. Within the Detailed display, you may choose to view listed items such as Clinical Features, and Biochemical Features.

Other roles of Urea Cycle intermediates:

The complete Urea Cycle occurs significantly only in liver. However some enzymes of this pathway are expressed in other cells and tissues, where they function to generate arginine and ornithine, which are precursors for other important molecules. For example, Argininosuccinate Synthase (see above), which catalyzes synthesis of the precursor to arginine, is found in most tissues. A mitochondrial enzyme Arginase II, distinct from the cytosolic Arginase involved in the Urea Cycle, cleaves arginine to yield ornithine.

Arginine, in addition to being a constituent of proteins, serves as precursor for synthesis of creatine and nitric oxide.

Nitric oxide (·NO) is a short-lived signal molecule with diverse roles in different cell types, including regulation of smooth muscle contraction, gene transcription, metabolism, and neurotransmission. Many of the effects of ·NO arise from its activation of a soluble cytosolic Guanylate Cyclase enzyme that catalyzes synthesis of cyclic-GMP (analogous in structure to cyclic-AMP).

Polyamines include putrescine, spermidine, and spermine. Ornithine is a major precursor for synthesis of polyamines. Conversion of ornithine to putrescine is catalyzed by Ornithine Decarboxylase.

The cationic polyamines have diverse roles in cell growth & proliferation. Disruption of polyamine synthesis or metabolism leads to disease in animals & humans.

Polyamines putrescine & spermidine

Additional material on Amino Acid Catabolism (N):
Readings, Test Questions & Tutorial