Catalysis of the Hydrolysis of Phosphorylated Pyridines by Alkaline Phosphatase

Bacterial alkaline phosphatase is an active catalyst for the hydrolysis of N-phosphorylated pyridines, with values of the second-order rate constant kmt/Km in the range 0.4-1.2 X lo6 M-1 s-l a t pH 8.0, 25 OC. There is little or no dependence of the rate on the pKa of the leaving group; the value of @le is 0 f 0.05, which may be compared with @le = -1 .O 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 a t low substrate concentrations. Alkaline phosphatase is a highly efficient catalyst for the hydrolysis of phosphate esters and other phosphate derivatives (McComb et al., 1979). Each monomer of the dimeric enzyme contains two Zn2+ ions, which are presumably involved in catalysis of the formation and hydrolysis of a phosphorylated serine hydroxyl group at the active site during turnover of the enzyme (Engstrom, 1961; Schwartz & Lipmann, 1961). The three-dimensional structure of the enzyme has been determined by X-ray crystallography (Sowadski et al., 1985; Kim & Wyckoff, 1991). It is difficult to probe the catalytic mechanism of alkaline phosphatase by steady-state kinetics because k,,, represents either rate-limiting hydrolysis of the phosphoenzyme intermediate, at pH < 7, or rate-limiting dissociation of inorganic phosphate from the enzyme, at pH > 7 (Reid & Wilson, 1971; Bloch & Schlesinger, 1973; Baleet al., 1980). However, the second-order rate constant for reaction of the enzyme with substrate, k,a,/Km, is a measure of the first irreversible step of the reaction and might provide information about the catalytic process. Hall and Williams (1986) have reported that the values of k,t/Km for the hydrolysis of a series of substituted phenyl phosphate monoesters show only a small dependence on the PKa of the leaving group, with a value of j31g = -0.19 from a Bronsted-type correlation of log k,t/Km against the PKa of the leaving group. It has been suggested that general acid-base catalysis may contribute to the observed catalysis (Sowadski et al., 1985) and that a small dependence of the rate on the structure of the leaving group could be caused by protonation of the leaving oxygen atom by an acidic group or coordination with a metal ion at the active site of the enzyme in the transition state (Williams et al., 1973; Hall & Williams, 1986). This electrophilic assistance could facilitate leaving group departure and decrease the development of negative charge on the leaving group, so that only a small dependence of the rate on leaving group structure would be observed. An increase in j31r from -1.2 to -0.7 in the presence t Contribution No. 1754 from the Graduate Department of Biochemistry, Brandeis University, Waltham, MA 02254-91 10. This research was supported in part by grants from the National Institutes of Health (GM-20888) and the National Science Foundation (DMB-8715832). Current address: Department of Biochemistry, B400 Beckman Center, Stanford University, Stanford, CA 94305-5307. 0006-2960/93/0432-8137$04.00/0 of Zn2+ ions has been observed for the nonenzymatic hydrolysis of substituted salicyl phosphates (Steffens et al., 1975). However, other possible explanations for this small dependence on leaving group structure include an electrostatic interaction with a cationic group at the active site that offsets the development of negative charge on oxygen without coordination, a partially rate-limiting conformational change, or rate-limiting productive binding of substrate to the enzyme, which could be diffusion-controlled (Trentham & Gutfreund, 1968; Fernley & Walker, 1969; Hall & Williams, 1986). We were interested in examining further the possibility that protonation of the leaving group in the transition state is responsible for the small dependence on leaving group structure, because this proton transfer provides a mechanism that could contribute to the catalysis that is brought about by the enzyme. For this reason, we have examined the values of k,,/Km for catalysis of the hydrolysis of a series of N-phosphorylated pyridines by alkaline phosphatase. These substrates, in contrast to phosphate esters and N-phenylphosphoramidates (Williams & Naylor, 197 1; Snyder & Wilson, 1972), do not have lone pair electrons on the leaving group that are available to accept a proton from an acid catalyst or to coordinate with a metal ion in the transition state. Therefore, the dependence of k,,,/K,,, on the structure of the leaving pyridine for the enzyme-catalyzed hydrolysis of these substrates provides a measure of the effect of leaving group structure on the rate of the catalyzed reaction in the absence of proton transfer or coordination with a metal ion. MATERIALS AND METHODS Materials. Alkaline phosphatase from Escherichia coli, type III-S from Sigma, was used without further purification. 4-Picoline, 3-picoline, 3,4-lutidine, and 3,5-lutidine were distilled, and 4-morpholinopyridine and 4-(dimethylamino)pyridine were recrystallized prior to phosphorylation. Phosphorylated pyridines were prepared as described previously (Skoog & Jencks, 1984; Herschlag & Jencks, 1987); 4-morpholinopyridine was a gift from Mark Skoog. The disodium salt of 4-nitrophenyl phosphate was recrystallized prior to use. Phosphorus oxychloride (Fisher) and CHES buffer (Sigma) were used without additional purification.