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:

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
pyruvate + NH4+

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

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. 

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]:

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

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. 

The amino acid arginine, in addition to being a constituent of proteins and an intermediate of the Urea Cycle, serves as precursor for synthesis of creatine and the signal molecule nitric oxide.
Synthesis of the radical species nitric oxide (NO) from arginine is catalyzed Nitric Oxide Synthase, a distant relative of cytochrome P450. Different isoforms of Nitric Oxide Synthase (e.g., eNOS expressed in endothelial cells and nNOS in neuronal cells) are subject to differing regulation.

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 regulatory 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).

Cytotoxic effects of NO observed under some conditions are attributed to its non-enzymatic reaction with superoxide (O2-) to form the strong oxidant peroxynitrite. (ONOO-).

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

There is no tRNA for citrulline, and this amino acid is not incorporated translationally into proteins. However, Ca++-activated Peptidylarginine Deiminases convert arginine residues within proteins to citrulline as a post-translational modification.

The substitution of citrulline, which lacks arginine's positive charge, may alter structure and properties such as binding affinities of a protein. For example, citrullination of certain proteins, including keratin intermediate filament proteins, is essential to terminal differentiation of skin cells.

Excessive protein citrullination, with production of antibodies against citrullinated proteins, is found to be a factor in the autoimmune diseases such as rheumatoid arthritis and multiple sclerosis.

Copyright 1998-2008 by Joyce J. Diwan. All rights reserved.

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

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