Mechanistic studies of protein tyrosine phosphatases YopH and Cdc25A with m-nitrobenzyl phosphate

Daniel F. McCain, Piotr K. Grzyska, Li Wu, Alvan C. Hengge, Zhong Yin Zhang

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18 Scopus citations

Abstract

Protein tyrosine phosphatases (PTPs) constitute a large family of signaling enzymes that include both tyrosine specific and dual-specificity phosphatases that hydrolyze pSer/Thr in addition to pTyr. Previous mechanistic studies of PTPs have relied on the highly activated substrate p-nitrophenyl phosphate (pNPP), an aryl phosphate with a leaving group pKa of 7. In the study presented here, we employ m-nitrobenzyl phosphate (mNBP), an alkyl phosphate with a leaving group pKa of 14.9, which mimics the physiological substrates of the PTPs. We have carried out pH dependence and kinetic isotope effect measurements to characterize the mechanism of two important members of the PTP superfamily: Yersinia PTP (YopH) and Cdc25A. Both YopH and Cdc25A exhibit bell-shaped pH-rate profiles for the hydrolysis of mNBP, consistent with general acid catalysis. The slightly inverse 18(V/K)nonbridge isotope effects (0.9999 for YopH and 0.9983 for Cdc25A) indicate a loose transition state with little nucleophilic participation for both enzymes. The smaller 18(V/K)bridge primary isotope effects (0.9995 for YopH and 1.0012 for Cdc25A) relative to the corresponding isotope effects for pNPP hydrolysis suggest that protonation of the leaving group oxygen at the transition state by the general acid is ahead of P-O bond fission with the alkyl substrate, while general acid catalysis of pNPP by YopH is more synchronous with P-O bond fission. The isotope effect data also confirm findings from previous studies that Cdc25A utilizes general acid catalysis for substrates with a leaving group pKa of > 8, but not for pNPP. Interestingly, the difference in the kinetic isotope effects for the reactions of aryl phosphate pNPP and alkyl phosphate mNBP by the PTPs parallels what is observed in the uncatalyzed reactions of their monoanions. In these reactions, the leaving group is protonated in the transition state, as is the case in PTP-catalyzed reactions. Also, the phosphoryl group in the transition states of the enzymatic reactions does not differ substantially from those of the uncatalyzed reactions. These results provide further evidence that these enzymes do not change the transition state but simply stabilize it.

Original languageEnglish (US)
Pages (from-to)8256-8264
Number of pages9
JournalBiochemistry
Volume43
Issue number25
DOIs
StatePublished - Jun 29 2004

ASJC Scopus subject areas

  • Biochemistry

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