Humboldt State University ® Department of Chemistry

Richard A. Paselk

Chem 432


Spring 2009

Lecture Notes: 6 February

© R. Paselk 2006


Nitrogen Metabolism

Protein catabolism and anabolism are often out of sync - either no additional protein is needed or the amino acid composition of the synthesized proteins is not identical to the protein being hydrolyzed. Neither protein nor amino acids are stored as such. Thus organisms must frequently degrade excess amino acids. Two paths may be available: deaminate the unneeded amino acids and breakdown the carbon skeletons for energy or for storage as fat or carbohydrate and eliminate the nitrogen; or the nitrogen may be transferred to another carbon backbone to make a needed amino acid.

Nitrogen can be eliminated in a variety of forms, depending on the physiological conditions experienced by the organism:


Amino Acid Deamination

For any of these products nitrogen must first be removed from the amino acids. Ammonia is often an intermediate if not the final product in all of these organisms. Most ammonia results from the deamination of a single amino acid: glutamate, via the Glutamate dehydrogenase reaction:

Structural diagram of the Glutamate DH reaction

This enzyme is somewhat unusual in that it can use either NADH or NADPH. Glutamate DH is activated by ADP and inhibited by GTP in vitro so they may regulate it in vivo. Of course these nucleotides would not provide a highly sensitive regulation, activity would only loosely follow energy charge.

How are the other amino acids deaminated? Most are transaminated, transferring their N to make glutamate:

amino acid + alpha-ketoglutarate equilibrium double arrow alpha-keto acid + glutamate

Transamination of Amino Acid

There are three main transaminases or Amino transferases, all requiring Pyridoxal-P as a cofactor:

The various aminotransferases in the liver all funnel excess N to glutamate and aspartate. Glutamate can then be deaminated by Glutamate DH to give ammonia, contributing up to 1/2 of the N in urea. Aspartate provides N directly to urea synthesis, contributing 1/2 of urea N.

Let's look at the aminotransferase reaction and the cofactor pyridoxal-phosphate. Pyridoxal-P (PLP) is derived from vitamin B6 (pyridoxine) via phosphorylation. Vitamin B6 is thus essential for protein metabolism.

structural diagrams of Pyridoxine and Pyridoxal-P

Pyridoxal-P is used to form Schiff bases with its various substrates, but in this case the nitrogen is provided by the substrate. PLP acts as an electrophilic catalyst; since none of the amino acids are electrophilic, cofactors must be provided when electrophilic covalent catalysis is needed.

As we saw before the transaminases catalyze symmetrical reactions:

amino acid + 2-oxoglutarate 2-oxoacid + glutamate

An amino acid reacts with a keto acid to give an amino acid and a keto acid. Thus one might expect a symmetrical mechanism, which is in fact the case. In the diagram below only the first half of the reaction mechanism is shown. The second half simply "reflects" the first half. It involves substituting the second keto acid in the right-hand step and reversing the process to take the second amino acid off in the left-hand step to complete the reaction (change the R s to R' s in the reverse steps):

Structural diagram of the chemical mechanism of Transaminase

Note that the PLP starts out covalently linked to a lysine-N, which is displaced by the reactant amino acid. During the remainder of the reaction the PLP is held non-covalently in the active site. In the final step the lysine amino group on the enzyme displaces the product amino acid nitrogen to release the free amino acid.

Chemically the reactant amino acid is oxidized (aldimine-ketimine shift in the mechanism) to release the keto acid product. The keto acid reactant is then reduced (ketimine-aldimine shift) to release the amino acid product. As might be expected the enzyme exhibits Ping-Pong Bi Bi kinetics:

"Kinetic mechanism diagram for the Ping-Pong Bi Bi kinetics of Transaminase

Pathway Diagrams

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Lecture Notes

Last modified 6 February 2009