SUMMARY Bovine Glutamate Dehydrogenase is potently inhibited by zinc and the major impact is on Vmax suggesting a V-type effect on catalysis or product release. Zinc inhibition decreases as glutamate concentrations decrease suggesting a role for subunit interactions. With the monocarboxylic amino acid, norvaline, which gives no evidence of subunit interactions zinc does not inhibit. Zinc significantly decreases the size of the pre-steady state burst in the reaction but does not affect NADPH binding in the Enzyme-NADPH-glutamate complex that governs the steady state turn-over again suggesting that zinc disrupts subunit interactions required for catalytic competence. While differential scanning calorimetry suggests zinc binds and induces a slightly conformationally more rigid state of the protein, limited proteolysis indicate regions in the vicinity of the antennae regions and the trimer-trimer interface become more flexible. The structures of GDH bound with zinc and europium show that zinc binds between the three dimers of subunits in the hexamer, a region shown to bind novel inhibitors that block catalytic turnover and is consistent with the above findings. In contrast, europium binds to the base of the antenna region and appears to abrogate the inhibitory effect of zinc. Structures of various states of the enzyme have shown that both regions are heavily involved in the conformational changes associated with catalytic turnover. These results suggest that the V-type inhibition produced with glutamate as the substrate results from disruption of subunit interactions necessary for efficient catalysis rather than by a direct effect on the active site conformation.

Zinc is a potent inhibitor of higher eukaryotic glutamate dehydrogenases. The mechanism of inhibition has previously been investigated using initial rate kinetic studies with either the dicarboxylic amino acid glutamate 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 goals of the current study were to quantitate the zinc binding, identify potential binding sites and determine whether or not zinc binding may be a] close to one of the three tryptophans in the protein, and b] induce conformational effects in the protein. Using the zinc sensitive chromophore 4‐par it is shown that zinc binds to glutamate dehydrogenase with a 1:1 stoichiometry with respect to polypeptide chains. Using tryptophan fluorescence spectroscopy it appears that zinc affects the most hydrophobic of the three tryptophans in the protein. Examination of the three dimensional structure of the enzyme raises the possibility that zinc binds at the subunit interface in the “twisty tie” region of the molecule. This is consistent with both the location of the most hydrophobic tryptophan and the fact that zinc appears to inhibit by disrupting subunit interactions necessary for full activity with glutamate as substrate.Supported by NSF grant MCB 0448905 to EB

Zinc is a potent inhibitor of higher eukaryotic glutamate dehydrogenase but the mechanism of inhibition has not been established. The goal of the current study is to correlate zinc binding under various conditions with changes in activity and conformational flexibility. Using Isothermal Titration Calorimetry it is shown that zinc binds to glutamate dehydrogenase with a 1:1 stoichiometry in the presence or absence of amino acid substrates. Using tryptophan fluorescence spectroscopy it appears that zinc affects the most hydrophobic of the three tryptophans in the protein. Examination of the three dimensional structure of the enzyme suggests that zinc may bind at the subunit interface in the "twisty tie" region of the molecule. This is consistent with both the location of the most hydrophobic tryptophan and the fact that zinc appears to inhibit by disrupting subunit interactions necessary for full activity with glutamate as substrate. Differential Scanning Calorimetry and Limited Proteolysis approaches demonstrate that zinc binding induces changes in the conformational flexibility of glutamate dehydrogenase which could be responsible for this effect.This research is supported by a Grant from the National Science Foundation: MCB 0448905 to EB