Probing the transition-state structure of dual-specificity protein phosphatases using a physiological substrate mimic

Biochemistry
Piotr K GrzyskaJ M Denu

Abstract

Dual-specificity phosphatases (DSPs) belong to the large family of protein tyrosine phosphatases that contain the active-site motif (H/V)CxxGxxR(S/T), but unlike the tyrosine-specific enzymes, DSPs are able to catalyze the efficient hydrolysis of both phosphotyrosine and phosphoserine/threonine found on signaling proteins, as well as a variety of small-molecule aryl and alkyl phosphates. It is unclear how DSPs accomplish similar reaction rates for phosphoesters, whose reactivity (i.e., pK(a) of the leaving group) can vary by more than 10(8). Here, we utilize the alkyl phosphate m-nitrobenzyl phosphate (mNBP), leaving-group pK(a) = 14.9, as a physiological substrate mimic to probe the mechanism and transition state of the DSP, Vaccinia H1-related (VHR). Detailed pH and kinetic isotope effects of the V/K value for mNBP indicates that VHR reacts with the phosphate dianion of mNBP and that the nonbridge phosphate oxygen atoms are unprotonated in the transition state. (18)O and solvent isotope effects indicate differences in the respective 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 s...Continue Reading

References

Aug 22, 2002·Journal of the American Chemical Society·Dilipkumar AsthagiriDonald Bashford
Oct 23, 2003·Journal of the American Chemical Society·Piotr K GrzyskaAlvan C Hengge

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Citations

Jan 26, 2006·Journal of the American Chemical Society·Jesse G Zalatan, Daniel Herschlag
Jul 2, 2009·The Journal of Biological Chemistry·Christopher A Bonham, Panayiotis O Vacratsis
Aug 10, 2006·Chemical Reviews·W Wallace Cleland, Alvan C Hengge
Apr 30, 2020·Biochemistry·Victor A BeaumontJ Patrick Loria

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