|Lecture Notes: 19 April||
Last time we looked at the Glutamate dehydrogenase 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:
Transamination of Amino Acids: 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.
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):
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:
Pyridoxal-P catalyzes a variety of other reactions involving amino acids involving destabilization of bonds to the alpha-carbon:
In addition, Pyridoxal-P catalyzes the direct deamination of serine and threonine via the destabilization of the side chain hydroxyl group. This reaction is favored by the excellent leaving group (water) on the beta-carbon. Thus PLP catalyzes the removal of water in Serine dehydratase:
The resulting aminoacrylate is then hydrated to give pyruvate as the product. The analogous reaction is catalyzed by Threonine dehydratase to give 2-oxobutyrate.
Other, non PLP catalyzed, direct deaminations include the hydrolysis of the amide nitrogens of glutamine and asparagine, and the deamination of histidine by histidase to give urocanate and ammonium.
L- & D-Amino acid oxidases are flavoproteins which catalyze the direct oxidation of amino acids in what can be considered detoxification reactions. The aa oxidases exhibit broad specificities. These enzymes occur in the peroxisomes of liver and kidney, where the hydrogen peroxide produced can be eliminated by catalase without damaging the cell:
Note here that unlike mixed function oxidases we are going to peroxide instead of water, and oxygen in peroxide has an oxidation state of -1, so only two electrons are required. So this reaction is not energy intensive - no NADH required, no ATP equiv. lost. But a very toxic product, hydrogen peroxide is produced! (Generally done in isolation in organelles such as peroxisomes which contain catalase to destroy peroxide.)
Last modified 19 April 2007