Catalysis of the Hydrolysis of Phosphorylated Pyridines by Mg(OH)+: A Possible Model for Enzymatic Phosphoryl Transfert

The second-order rate constants for reaction of the Mg2+ complexes of phosphorylated pyridine monoanions with Mg(OH)+ a re 104-106-fold larger than the second-order rate constants for their reaction with water (25 OC, ionic strength 1.5). Of the 106-fold rate enhancement with the phosphorylated 4morpholinopyridine/Mg2 complex, 1 04-fold is attributed to the greater nucleophilicity of Mg(OH)+ compared with water. The remaining catalysis of 102-fold is attributed to induced intramolecularity from positioning of the hydroxide ion and phosphoryl group by the Mg2+ ions. This reaction may provide a model for the role of a metal ion in increasing the concentration of the anions of enolpyruvate and serine and holding the nucleophile in the correct position for phosphoryl transfer in the reactions catalyzed by pyruvate kinase and alkaline phosphatase, for example. Some mechanisms that can provide catalysis of phosphoryl transfer through a metaphosphate-like transition state a re reviewed briefly. %e mechanisms for catalysis of phosphoryl transfer reactions by enzymes and the role of Mg2+ in this catalysis are not well understood. Two mechanisms that can contribute to this catalysis are (1) lowering the pK, of water, which allows deprotonation and formation of the stronger nucleophile, Mg(OH)+, a t physiological p H values and (2) providing a template to hold the reactants in place in the transition state (Lowenstein, 1958; Jencks, 1969; Spiro, 1973; Cooperman, 1976; Benkovic & Schray, 1978; Knowles, 1981). In this paper we show that Mg2+ catalyzes phosphoryl transfer from pyridines (Chart I) to water by these mechanisms. Although the catalysis is not large, the reaction may serve as a model for the role of a metal ion in catalysis by enzymes such as pyruvate kinase and alkaline phosphatase. It is suggested that this is one of several mechanisms that can provide significant catalysis of phosphoryl transfer, even through a transition state that is metaphosphate like. Several examples of rapid phosphoryl transfer to metal hydroxides have been reported [Lipkin et a]., 1959; Bamann & Trapmann, 1959; Bruice & Benkovic, 1966; Osterheld, 1972; Cooperman, 1976; Milburn et al. (1 985) and references cited therein]. However, many of these are two-phase systems that have been difficult to characterize quantitatively [e.g., Butcher and Westheimer (1955) and Jenkins (1988)l. Ambiguity of analysis because of uncertainty in the geometry for chelation of the bound metal ion has been circumvented in some reactions by the use of Co(II1) and other exchange-inert metal ions; several phosphoryl-transfer reactions to the hydroxide ion ligand of a Co(III)/substrate complex have been shown to give large rate enhancements, compared with hydrolysis of the free substrate (Farrell et al., 1969; Jones et al., 1983, 1984; Meyer & Cornelius, 1984; Haight et al., 1985; Milburn et al., 1985, and references cited therein). In this paper we describe an analysis of the factors that contribute to catalysis by Mg(OH)+ in homogeneous solution. MATERIALS A N D METHODS Materials. y-Picoline and pyridine were purified by distillation. Aqueous solutions of phosphorylated 4'Contribution No. 1707. Chart I