ACTIVE-SITE MUTAGENESIS OF ESCHERICHIA-COLI ALKALINE-PHOSPHATASE - REPLACEMENT OF SERINE-102 WITH NONNUCLEOPHILIC AMINO-ACIDS

The active-site nucleophile in bacterial alkaline phosphatase, Ser-102, was replaced using site-directed mutagenesis, with leucine and, separately, alanine. Remarkably, the mutant enzymes S102A and S102L, which lack a nucleophilic side chain at position 102, still catalyze the hydrolysis of phosphate monoesters. The k(cat) values are, respectively, approximately 1/1000 and approximately 1/500 of the wild-type enzyme. The mutant enzymes have K(m) values, K(i) values (inorganic phosphate), and pH profiles that are similar to wild-type. Several experiments argue against the possibility of wild-type contamination. The S102L mutant has a substrate-dependent partition ratio which is consistent with a change in the enzyme mechanism. Direct hydrolysis by zinc-activated water may account for the catalytic activity of these mutant enzymes. This work illustrates the use of site-directed mutagenesis to uncover latent enzyme activity and reinforces the idea that the zinc atoms in the active site are the critical structural feature in the design of alkaline phosphatase.