Glucose 6-Phosphate Dehydrogenase from Leuconostoc mesenteroides: KINETIC STUDIES

Abstract

Glucose 6-phosphate dehydrogenase from Leuconostoc mesenteroides catalyzes the oxidation of glucose 6-phosphate by either NADP+ or NAD+. Steady state initial velocity studies and product inhibition studies using NADPH were conducted for the NADP-linked reaction catalyzed by this enzyme. The data were consistent with a simple, ordered, sequential mechanism for this reaction in which NADP+ is bound first to the enzyme and NADPH released last. Kinetic studies of the NAD-linked reaction indicated a more complex mechanism. Initial velocity studies, product, and alternate product inhibitions using NADH and NADPH, respectively, and alternate substrate studies using either NAD+ and NADP+ with glucose 6-phosphate, or glucose 6-phosphate and 2-deoxyglucose 6-phosphate with NAD+, suggested an ordered, sequential mechanism with isomerization of free enzyme. The enzyme form which binds NADH is proposed to be the same form binding NADP+ and NADPH; NAD+ is assumed to bind to an isomeric form. A detailed study of the effect of varying pH from 5.74 to 9.90 was carried out for the NAD-linked reaction. From the changes in kinetic constants with pH some tentative suggestions emerged concerning enzyme groups which may participate in the binding of NAD+ and glucose 6-phosphate. Among these, the most clear-cut was the participation of a group on the enzyme with pK' = 8.9 in binding of glucose 6-phosphate. Evidence was obtained that this group is not a cysteine. Further evidence, using pyridoxal 5'-phosphate, suggested that the group may be the e-NH2 group of a lysine.