Transition state and rate-limiting step of the reaction catalyzed by the human dual-specificity phosphatase, VHR.
The dual-specificity phosphatases are unusual catalysts in that they can utilize protein substrates containing phosphotyrosine as well as phosphoserine/threonine. The dual-specificity phosphatases and the protein-tyrosine phosphatases (PTPases) share the active site motif (H/V)C(X)5R(S/T), but display little amino acid sequence identity outside of the active site. Although the dual-specificity phosphatases and the PTPases appear to bring about phosphate monoester hydrolysis through a similar mechanism, it is not clear what causes the difference in the active-site specificity between the two groups of enzymes. In this paper, we show that the human dual-specificity phosphatase, VHR [for VH1-Related; Ishibashi et al. (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 12170-12174], is rather promiscuous toward small phosphate monoesters (including both aryl and alkyl phosphates of primary alcohols) with effectively identical kcat/Km and kcat values while the pKa values of the leaving groups (phenols or alcohols) varied from 7 to 16. Linear free-energy relationship analysis of kcat and kcat/Km of the enzyme-catalyzed hydrolysis reaction suggests that a uniform mechanism is utilized for both the aryl and alkyl substrates. The very small dependency of kcat/Km on the leaving group pKa can be accounted for by the protonation of the leaving group. Pre-steady-state burst kinetic analysis of the VHR-catalyzed hydrolysis of p-nitrophenyl phosphate provides direct kinetic evidence for the involvement of a phosphoenzyme intermediate in the dual specificity phosphatase-catalyzed reaction. The rate-limiting step for the VHR-catalyzed hydrolysis of p-nitrophenyl phosphate corresponds to the decomposition of the phosphoenzyme intermediate.(ABSTRACT TRUNCATED AT 250 WORDS)
Department of Molecular Pharmacology, Albert Einstein College of Medicine, Bronx, New York 10461, USA.
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Zhang
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Wu
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Chen
D000595:Amino Acid Sequence; D054637:Dual-Specificity Phosphatases; D006801:Humans; D007700:Kinetics; D008962:Models, Theoretical; D008969:Molecular Sequence Data; D010768:Phosphoserine; D010769:Phosphothreonine; D019000:Phosphotyrosine; D017027:Protein Tyrosine Phosphatases / Q000737:chemistry / Q000378:metabolism*; D013379:Substrate Specificity
D010769:Phosphothreonine; D010768:Phosphoserine; D019000:Phosphotyrosine; C489855:dual specificity phosphatase 12; D054637:Dual-Specificity Phosphatases; D017027:Protein Tyrosine Phosphatases