A bifunctional enzyme, fructose-6-phosphate, 2-kinase:fructose-2,6-bisphosphatase, catalyzes synthesis and hydrolysis of fructose 2,6-bisphosphate. The phosphatase reaction occurs in two steps: The formation of a phosphoenzyme intermediate and release of β-D-fructose 6-phosphate, followed by hydrolysis 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 kcat for the wild-type enzyme exhibits pK values of 5.6 and 9.1. The pH dependence of kcat for the E325A mutant enzyme gives an 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 acids tested, are the most potent activators. The kcat vs pH profile of the 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 serving 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 phosphoenzyme intermediate, rescuing some of the activity lost upon eliminating the glutamate side chain.
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