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

Abstract 6-Phosphogluconate dehydrogenase (EC 1.1.1.44) has been purified from Cryptococcus 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 , and K m values of 13 μ M for 6-phosphogluconate and 0.89 μ M for NADP. This enzyme, like many fungal dehydrogenases, was inhibited by Zn 2+ , with the inhibition pattern being competitive versus the nonnucleotide substrate. In the presence of micromolar Zn 2+ , 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: k 6 , 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*Zn 2+ to E + Zn 2+ was 0.021 μ M .