DEPENDENCE OF THE PHOSPHORYLATION OF ALKALINE-PHOSPHATASE BY PHOSPHATE MONOESTERS ON THE PK(A) OF THE LEAVING GROUP

The hydrolysis and transphosphorylation reactions of a series of phosphate monoesters, ROPO(3)(2-) (R = 2,4-dinitrophenyl, 4-nitrophenyl, phenyl, glucose-1, glycerol-1, methyl, ethyl, and dodecyl), catalyzed by Escherichia coli alkaline phosphatase and a mutant enzyme, Ser102Cys, have been studied at alkaline pH using the rates of change in the P-31 NMR signals of substrate, the hydrolysis product (inorganic phosphate), and the transphosphorylation product (O-Tris phosphate) as the assay. The k(cat) at pH 8.0 for the wild-type enzyme is similar to 30 s(-1) and is independent of the nature of the R group, when the pK(a) of the leaving group is <10. Under these conditions the rate of phosphorylation is much faster than dissociation of inorganic phosphate, 15-60 s(-1). If the pK(a) of the leaving group is between 10 and 15, phosphorylation and dissociation of the product phosphate both contribute to the rate limit. If the pK, of the leaving group is >15, phosphorylation is rate limiting. A Bronsted plot of log k(cat) vs pK(a) of the leaving group for those substrates for which phosphorylation is rate limiting yields a beta(1g) of similar to-0.6. In contrast to the wild-type enzyme, the log k(cat) values for the S102C mutant enzyme catalyzing the hydrolysis of phosphate esters are linearly dependent on the pK(a)'s of the leaving group throughout the range of pK(a) from 4 to 16. Phosphorylation of C102 is the rate controlling step, and k(cat) is independent of the Tris concentration as predicted for rate limiting phosphorylation. The Bronsted constant, beta(1g), is similar to-0.3. The catalytic rate for the S102C mutant is at least 50-fold slower than that for the wild-type enzyme. The dependence of both k(cat) and the beta(1g), value on the nature of the nucleophile suggests that phosphorylation of the enzyme is primarily associative in character.