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 phosp hate monoesters, ROPO(3)(2-) (R = 2,4-dinitrophenyl, 4-nitrophenyl, ph enyl, 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-3 1 NMR signals of substrate, the hydrolysis product (inorganic phosphat e), and the transphosphorylation product (O-Tris phosphate) as the ass ay. 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 phosphory lation 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, phospho rylation and dissociation of the product phosphate both contribute to the rate limit. If the pK, of the leaving group is >15, phosphorylatio n is rate limiting. A Bronsted plot of log k(cat) vs pK(a) of the leav ing group for those substrates for which phosphorylation is rate limit ing 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 t he 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. Pho sphorylation of C102 is the rate controlling step, and k(cat) is indep endent of the Tris concentration as predicted for rate limiting phosph orylation. The Bronsted constant, beta(1g), is similar to-0.3. The cat alytic 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(1 g), value on the nature of the nucleophile suggests that phosphorylati on of the enzyme is primarily associative in character.