Slow-Binding Inhibition of 6-Phosphogluconate Dehydrogenase by Zinc Ion

Abstract

6-Phosphogluconate dehydrogenase (EC 1.1.1.44) has been purified fromCryptococcus neoformans,an encapsulated yeast that is an opportunistic pathogen of AIDS patients. The dimeric enzyme had a subunit molecular weight of 50,000, a specific activity of 50 units mg−1, andKmvalues of 13 μMfor 6-phosphogluconate and 0.89 μMfor NADP. This enzyme, like many fungal dehydrogenases, was inhibited by Zn2+, with the inhibition pattern being competitive versus the nonnucleotide substrate. In the presence of micromolar Zn2+, the reaction was biphasic, with the reduction of NADP proceeding initially at the control rate, but, over the time course of 20–300 s, this initial nonlinear phase reached a final, linear steady state with a slower velocity. This pattern is indicative of a slow binding inhibition process, for which we have calculated the following kinetic constants:k6, the limiting rate constant for the transition from initial to final steady state was 0.0024 s−1, corresponding to a half-time of 300 s;K*i, the overall equilibrium constant for the dissociation of E*Zn2+to E + Zn2+was 0.021 μM.