CATALYSIS OF THE HYDROLYSIS OF PHOSPHORYLATED PYRIDINES BY ALKALINE-PHOSPHATASE HAS LITTLE OR NO DEPENDENCE ON THE PK(A) OF THE LEAVING GROUP

Bacterial alkaline phosphatase is an active catalyst for the hydrolysis of N-phosphorylated pyridines, with values of the second-order rate constant k(cat)/K(m) in the range 0.4-1.2 x 10(6) M-1 s-1 at pH 8.0, 25-degrees-C. There is little or no dependence of the rate on the pK(a) of the leaving group; the value of beta(lg) is 0 +/- 0.05, which may be compared with beta(lg)=-1.0 for the nonenzymic reaction. Phosphorylated pyridines do not have a free electron pair available for protonation or coordination of the leaving group. Therefore, this result means that the similar, small dependence on leaving group structure for the enzyme-catalyzed hydrolysis of phosphate esters [Hall, A. D., & Williams, A. (1986) Biochemistry 25, 4784-4790) does not provide evidence for general acid catalysis or electrophilic assistance of leaving group expulsion. The results are consistent with the hypothesis that productive binding of the substrate, which may involve a conformational change, is largely rate limiting for turnover of the enzyme at low substrate concentrations.