Rate-determining step of Escherichia coli alkaline phosphatase altered by the removal of a positive charge at the active center

Escherichia coli alkaline phosphatase catalyzes both the nonspecific hydrol ysis of phosphomonoesters and a transphosphorylation reaction in which phos phate is transferred to an alcohol via a phosphoseryl intermediate. The rat e-determining step for the wild-type enzyme is pH dependent. At alkaline pH , release of the product phosphate from the noncovalent enzyme-phosphate co mplex determines the reaction rate, whereas at acidic pH hydrolysis of the covalent enzyme-phosphate complex controls the reaction rate. When the lysi ne at position 328 was substituted with a cysteine (K328C), the rate-determ ining step at pH 8.0 of the mutant enzyme was altered so that hydrolysis of the covalent intermediate became limiting rather than phosphate release. T he transphosphorylation activity of the K328C enzyme was selectively enhanc ed, while the hydrolysis activity was reduced compared to that of the wild- type enzyme. The ratio of the transphosphorylation to the hydrolysis activi ties increased 28-fold for the K328C enzyme in comparison with the wild-typ e enzyme. Several other mutant enzymes for which a positive charge at the a ctive center is removed by site-specific mutagenesis share this characteris tic of the K328C enzyme. These results suggest that the positive charge at position 328 is at least partially responsible for maintaining the balance between the hydrolysis and transphosphorylation activities and plays an imp ortant role in determining the rate-limiting step of E. coli alkaline phosp hatase.