Mechanisms of Hydrolysis of O-Phosphorothioates and Inorganic Thiophosphate by Escherichia coli Alkaline Phosphatase

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Abstract Hydrolyses of phosphate and O-phosphorothioate monoesters by Zn(II) and Co(II) alkaline phosphatases (Escherichia coli) are compared. Although the rate of hydrolysis of the O-phosphorothioate by the Zn(II) enzyme is ∼100-fold slower than the hydrolysis of phosphate esters, the kinetic data of the transient and steady state phases of the reaction show that the stepwise mechanisms are apparently identical. Reaction of the Zn(II) enzyme with O-phosphoro[35S]thioate at acid pH results in the incorporation of covalently linked 35S into the enzyme during the transient phase of the reaction, and H2S is not released. Thus a thiophosphoryl enzyme forms analogous to the phosphoryl enzyme formed by phosphate esters. In contrast, H2S and Pi are the products of hydrolysis of inorganic thiophosphate by the enzyme. The variation in the absolute and relative magnitudes of the rate constants describing (thio)phosphorylation and de-(thio)phosphorylation of the enzyme can be explained on the basis of the steric and electronic effects of sulfur substitution in retarding nucleophilic displacement processes involved in the generation and decomposition of reaction intermediates. Sulfur substitution reduces the negative homotropic interactions between the two active sites such that at concentrations of O-phosphorothioate g10-4 m a 2-mole burst of phenol is observed in the pre-steady state phase of the reaction. Enzyme-catalyzed hydrolysis of thiophosphate and solvent-phosphate -OH exchange appear to proceed via a rate-limiting, pH-independent phosphorylation. The catalytic rate of H2S release from thiophosphate is ∼10-fold greater than the rate of hydrolysis of O-phosphorothioate, but ∼50-fold less than the rate of hydrolysis of phosphate esters. Substitution of Co(II) for the Zn(II) of alkaline phosphatase increases the rate of O-phosphorothioate hydrolysis by 4-fold, but results in a 5-fold reduction in the rate of phosphate ester hydrolysis and a 10-fold drop in the rate of thiophosphate hydrolysis. While these variations are not fully explicable at present, they reflect differences in the rates of the same mechanistic steps, emphasizing the involvement of the metal ion in all processes required for catalytic hydrolysis.