The Effects of Zinc on Glutamate Dehydrogenase

Abstract

Glutamate Dehydrogenase consist of six identical subunits and subunit interactions are known to play significant roles in both catalysis and regulation. Zinc is a potent inhibitor of eukaryotic glutamate dehydrogenases. The mechanism of this inhibition as been investigated using initial rate kinetic studies with either the dicarboxylic amino acid glutamate as substrate or the mono carboxylic alternative substrate norvaline. With glutamate zinc potently inhibits with the apparent affinity for zinc decreasing as the glutamate concentration decreases. With norvaline no inhibition is seen. The effects of zinc on cofactor binding shows that zinc weakens the binding of the product NADPH. We have developed a competition binding assay using the chromagenic dye 4-PAR to show that zinc can bind to the enzyme with approximately equal affinity in the absence of the amino acid substrate or the presence of norvaline or glutamate. Norvaline, compared to glutamate is shown to greatly decrease the conformational flexibility of the protein indicating that the inhibitory effects of zinc require a dynamic structure of the protein. In stopped flow kinetic studies the effects of zinc on the burst amplitude indicate that zinc decreases the number of active sites in the hexamer available for catalysis. The ability of Norvaline to stabilize the enzyme and decrease it flexibility prevents subunit communication necessary for efficient catalysis and as a result the catalytic steps of the reaction are the overall rate limiting step. It is clear that zinc binds to the enzyme in the presence or absence of either substrate but it has no effect on catalysis in the reaction involving Norvaline. This research is supported by a Grant from the National Science Foundation: MCB 0448905 to EB