Metal-substrate interactions facilitate the catalytic activity of the bacterial phosphotriesterase

The bacterial phosphotriesterase from Pseudomonas diminuta is a zinc metalloenzyme which catalyzes the hydrolysis of a variety of organophosphorus nerve agents with high efficiency. The active site of the enzyme consists of a coupled binuclear metal center embedded within a cluster of histidine residues. Potential protein-substrate interactions at the active site were probed by a systematic variation of metal identity, leaving group potential, phosphate host, and amino acid replacement. In order to determine the roles of these metal ions in binding and catalysis, the microscopic rate constants and kinetic parameters were obtained with various divalent cations. The divalent cations that were utilized in this investigation consisted of Co2+, Ni2+, Cd2+, Zn2+, Mn2+, and the mixed-metal Zn2+/Cd2+ hybrid. The leaving group potential and phosphate host were varied by altering the pK(a) of the departing substituted phenol or thiophenol in either a diethyl phosphate or a diethyl thiophosphate substrate. The Bronsted plots for the nonenzymatic hydroxide catalyzed hydrolysis of these substrates showed a linear dependence between the pseudo-first-order rate constant and the pK(a) of the leaving group. Enzymatic activities of the wild-type enzyme with these same substrates varied by over 7 orders of magnitude over the entire experimental pK(a) range (4.1-10.3), and the corresponding Bronsted plots were nonlinear, Those substrates with leaving groups with high pK(a) values were limited by the rate of bond cleavage while those substrates having leaving groups with low pK(a) values were limited by a conformational change or binding event. Thiophosphate substrates having leaving groups with high pK(a) values were better substrates than the corresponding phosphate analogues. These results are consistent with the direct coordination of one or both metal ions with the phosphoryl sulfur or oxygen atom of the substrate. A large dependence of the rate on the leaving group rules out the possibility of protonation of the leaving group or electrostatic interaction of the leaving group oxygen (or sulfur) with a metal ion or cationic group at the active site. The large differences in the size of the beta(lg) over the range of metal ions utilized by the enzyme indicate that the metal ions polarize the phosphoryl group and alter the structure of the transition state. The values of V/K-m for the enzyme-catalyzed hydrolysis for a series of substituted thiophenol analogues were 10(2)-10(3)-fold smaller than those obtained for the hydrolysis of the corresponding phenolic substrates, suggesting that the bulkier sulfur substituent in the leaving group may induce conformational restrictions at the active site. With the zinc-substituted H201N mutant enzyme, there was a large decrease in the rate of phosphotriester hydrolysis but essentially no change in the rate of thiophosphotriester hydrolysis relative to the values observed for the zinc-substituted wild-type enzyme. These results suggest that a direct perturbation in the ligand structure of the binuclear metal center induces alterations in the mechanism of substrate hydrolysis.