4238
Biochemistry 1995,34, 4238-4245
Dependence of the Phosphorylation of Alkaline Phosphatase by Phosphate Monoesters on the PKa of the Leaving Group: Runyu Han and Joseph E. Coleman* Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, Connecticut 06511 Received August 22, 1994; Revised Manuscript Received December 23, 1994@
The hydrolysis and transphosphorylation reactions of a series of phosphate monoesters, ROP032(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, Serl02Cys, have been studied at alkaline pH using the rates of change in the 31P NMR signals of substrate, the hydrolysis product (inorganic phosphate), and the transphosphorylation product (0-Tris phosphate) as the assay. The kcatat pH 8.0 for the wild-type enzyme is -30 s-l and is independent of the nature of the R group, when the pK, of the leaving group is
