Kinetic studies of gluconate pathway enzymes from Schizosaccharomyces pombe

Abstract

Glucose dehydrogenase and gluconate kinase which catalyze two-step reactions of the gluconate pathway have been purified from Schizosaccharomyces pombe. Their steady-state kinetic studies were undertaken. The yeast glucose dehydrogenase requires NADP+ as an obligatory coenzyme and mediates the oxidation of D-glucose to D-gluconate via an ordered Bi Bi mechanism with NADP+ as the leading substrate. Kinetic constants for the dehydrogenase reactions have been measured. The yeast gluconate kinase requires Mg2+ as an activator. The phosphorylation catalyzed by the fission yeast gluconate kinase has been studied kinetically at a fixed concentration of Mg2+. The initial velocity and product inhibition results are consistent with a rapid equilibrium random Bi Bi mechanism with the formation of an abortive enzyme-ADP-gluconate complex. Dissociation constants of the two substrates, ATP and D-gluconate from various binary and ternary enzymic complexes, have been determined.