The mechanism of the phosphoryl transfer catalyzed by Yersiniaprotein-tyrosine phosphatase: a computational and isotope effect study

In order to evaluate various mechanistic proposals that have been made regarding the mechanism of the first step of the reaction catalyzed by protein-tyrosine phosphatases, new experimental data have been obtained, and some existing data have been carefully reevaluated. New kinetic isotope effect data for the uncatalyzed hydrolysis of p-nitrophenyl phosphate allow a better evaluation of previously reported data from enzymatic reactions with this substrate. The interpretation, and misinterpretation, of pH rate studies is considered. The pathway of this reaction has been modeled computationally and is found to be generally consistent with experimental studies, except for the extent of proton transfer to the leaving group

Isotope effects on enzymatic and nonenzymatic reactions of phosphorothioates

Introduction O-phosphorothioate analogs of phosphate monoesters have long been used to probe kinetic and stereochemical aspects of phosphoryl transfer. Both uncatalyzed reactions in solution and enzymatic reactions have been studied, the latter most notably with alkaline phosphatase. Large thio effects, defined as the ratio of the reaction rate with a phosphate substrate over that of the corresponding phosphorothioate, or k O /k S , observed in alkaline phosphatase have been used to infer an associative, triester-like mechanism Phosphate monoesters typically react in solution by mechanism A i

Isotope effects on enzymatic and nonenzymaticreactions of phosphorothioates

Kinetic isotope effects have been measured for the aqueous hydrolysis reactions of p-nitrophenyl phosphorothioate (pNPPT) and the diester ethyl p-nitrophenyl phosphorothioate, and for the alkaline phosphatase-catalyzed reaction with pNPPT. The results show that the transition states of the uncatalyzed reactions of the phosphorothioate mono- and diesters are very similar to those of the corresponding phosphate ester reactions. The secondary 18O nonbridge isotope effects in reactions of phosphate esters become more normal as the mechanism changes from dissociative, metaphosphate-like to associative, phosphorane-like. The opposite trend occurs in phosphorothioate esters, due to differences in the relative contributions of bond-order changes and bending modes to this isotope effect. The KIEs for the alkaline phosphatase-catalyzed reaction of pNPPT are most consistent with a tight, triester-like transition state, probably a result of perturbations resulting from the larger size of sulfur that lead to a nucleophile attack angle that is unfavorable for an in-line process with a loose transition state

Transition State Differences inHydrolysis Reactions of Alkyl versus Aryl Phosphate Monoester Monoanions

Although aryl phosphates have been the subject of numerous experimental studies, far less data bearing on the mechanism and transition states for alkyl phosphate reactions have been presented. Except for esters with very good leaving groups such as 2,4-dinitrophenol, the monoanion of phosphate esters is more reactive than the dianion. Several mechanisms have been proposed for the hydrolysis of the monoanion species. 18O kinetic isotope effects in the nonbridging oxygen atoms and in the P−O(R) ester bond, and solvent deuterium isotope effects, have been measured for the hydrolysis of m-nitrobenzyl phosphate. The results rule out a proposed mechanism in which the phosphoryl group deprotonates water and then undergoes attack by hydroxide. The results are most consistent with a preequilibrium proton transfer from the phosphoryl group to the ester oxygen atom, followed by rate-limiting P−O bond fission, as originally proposed by Kirby and co-workers in 1967. The transition state for m-nitrobenzyl phosphate (leaving group pKa 14.9) exhibits much less P−O bond fission than the reaction of the more labile p-nitrophenyl phosphate (leaving group pKa = 7.14). This seemingly anti-Hammond behavior results from weakening of the P−O(R) ester bond resulting from protonation, an effect which calculations have shown is much more pronounced for aryl phosphates than for alkyl ones

A nonhydrolyzable analogue ofphosphotyrosine, and related aryloxymethano- and aryloxyethano- phosphonic acids as motifs forinhibition of phosphatases

Nonhydrolyzable analogues of both stereoisomers of phosphotyrosine, and a series of related aryloxy (or thio) methyl and aryloxy (or thio) ethyl phosphonic acids of the general formula RX–(CH2)n–PO3H2 (where X = O or S and n = 1 or 2), have been tested as nonhydrolyzable mimetics of phosphatase substrates. These compounds were tested against a panel of phosphatases (two alkaline phosphatases, a protein–tyrosine phosphatase, and two serine/threonine phosphatases) with different active site motifs. The compounds exhibit competitive inhibition toward all enzymes tested, with the best inhibition expressed toward the Ser/Thr phosphatases. The stereoisomers of the phosphotyrosine analogues exhibited an unexpected difference in their inhibitory properties toward the protein–tyrosine phosphatase from Yersinia. The Ki for the d isomer is 33-fold lower than that of the l isomer, and is more than an order of magnitude lower than the reported Km of the substrate l-phosphotyrosine