Kinetic studies of the reactions catalyzed by glucose-6-phosphate dehydrogenase from Leuconostoc mesenteroides: pH variation of kinetic parameters.

The specificity and kinetic parameters of the reactions catalyzed by glucose-6-phosphate dehydrogenase from Leuconostoc mesenteroides has been examined under a range of conditions in order to elucidate details about the mechanism of action of this enzyme. The rate of oxidation of glucose 6-phosphate is inhibited by the addition of various organic solvents. However, the low, inherent glucose dehydrogenase activity of this enzyme was stimulated under these conditions, and was further activated by divalent anions that were observed to be inhibitors of the glucose 6-phosphate dehydrogenation. From an examination of the pH variation of the enzyme kinetic parameters two groups on the enzyme that appear to be involved in the binding of the phosphate group of the sugar substrate have been detected. An enzyme catalytic group, probably a carboxylic acid, has been identified that accepts the proton from the hydroxyl group at carbon-1 of the sugar substrate during its oxidation to a lactone. The ionization of a group on the enzyme with a pK of 8.7 resulted in an increase in the maximum velocity of the glucose-6-phosphate dehydrogenase activity of the enzyme as a consequence of a pH-dependent product release step that is no longer rate limiting at high pH. Stabilization of gluconic acid-delta-lactone against nonenzymatic hydrolysis by organic solvents has allowed the kinetic parameters of the reverse reaction to be reliably measured for the first time in a narrow pH range.