Glutamate 325 is a general acid-base catalyst in the reaction catalyzed byfructose-2,6-bisphosphatase

A bifunctional enzyme, fructose-6-phosphate, 2-kinase:fructose-2,6-bisphosp hatase, catalyzes synthesis and hydrolysis of fructose 2,6-bisphosphate. Th e phosphatase reaction occurs in two steps: the formation of a phosphoenzym e intermediate and release of beta -D-fructose 6-phosphate, followed by hyd rolysis of the phosphoenzyme. The objective of this study was to determine whether E325 in the Fru 2,6-Pase active site is an acid-base catalyst. The pH-rate profile for k(cat) for the wild-type enzyme exhibits pK values of 5 .6 and 9.1. The pH dependence of k(cat) for the E325A mutant enzyme gives a n increase in the acidic pK from 5.6 to 6.1. Formate, acetate, propionate, and azide accelerate the rate of hydrolysis of the E325A mutant enzyme, but not of the wild-type enzyme. Azide and formate, the smallest of the weak a cids tested, are the most potent activators. The k(cat) vs pH profile of th e E325A mutant enzyme in the presence of formate is similar to that of the wild-type enzyme. Taken together, these data are consistent with E325 servi ng an acid-base role in the phosphatase reaction. The exogenous low MW weak acids act as a replacement general base in the hydrolysis of the phosphoen zyme intermediate, rescuing some of the activity lost upon eliminating the glutamate side chain.