| Abstract
| - Dual-specificity phosphatases (DSPs) belong to the large family of protein tyrosine phosphatasesthat contain the active-site motif (H/V)CxxGxxR(S/T), but unlike the tyrosine-specific enzymes, DSPsare able to catalyze the efficient hydrolysis of both phosphotyrosine and phosphoserine/threonine foundon signaling proteins, as well as a variety of small-molecule aryl and alkyl phosphates. It is unclear howDSPs accomplish similar reaction rates for phosphoesters, whose reactivity (i.e., pKa of the leaving group)can vary by more than 108. Here, we utilize the alkyl phosphate m-nitrobenzyl phosphate (mNBP), leaving-group pKa = 14.9, as a physiological substrate mimic to probe the mechanism and transition state of theDSP, Vaccinia H1-related (VHR). Detailed pH and kinetic isotope effects of the V/K value for mNBPindicates that VHR reacts with the phosphate dianion of mNBP and that the nonbridge phosphate oxygenatoms are unprotonated in the transition state. 18O and solvent isotope effects indicate differences in therespective timing of the proton transfer to the leaving group and P−O fission; with the alkyl ester substrate,protonation is ahead of P−O fission, while with the aryl substrate, the two processes are more synchronous.Kinetic analysis of the general-acid mutant D92N with mNBP was consistent with the requirement ofAsp-92 in protonating the ester oxygen, either in a step prior to significant P−O bond cleavage or in aconcerted but asynchronous mechanism in which protonation is ahead of P−O bond fission. Collectively,the data indicate that VHR and likely all DSPs can match leaving-group potential with the timing of theproton transfer to the ester oxygen, such that diverse aryl and alkyl phosphoesters are turned over withsimilar catalytic efficiency.
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