Catalytic and chemical competence of regulation of Cdc25 phosphatase by oxidation/reduction

Jungsan Sohn, Johannes Rudolph

Research output: Contribution to journalArticlepeer-review

Abstract

Cdc25 phosphatases belong to the family of protein tyrosine phosphatases (PTPs) that contain an active-site cysteine and form a phosphocysteine intermediate. Recently, oxidation/reduction of activesite cysteines of PTPs, including Cdc25, has been proposed to serve as a form of reversible regulation for this class of enzymes. Here we provide in vitro evidence that supports the chemical and kinetic competence for oxidation/reduction of the active-site cysteines of Cdc25B and Cdc25C as a mechanism of regulation. Using kinetic measurements and mass spectrometry, we have found that the active-site cysteines of the Cdc25's are highly susceptible to oxidation. The rate of thiolate conversion to the sulfenic acid by hydrogen peroxide for Cdc25B is 15-fold and 400-fold faster than that for the protein tyrosine phosphatase PTP1B and the cellular reductant glutathione, respectively. If not for the presence of an adjacent (back-door) cysteine in proximity to the active-site cysteine in the Cdc25's, the sulfenic acid would rapidly oxidize further to the irreversibly inactivated sulfinic acid, as determined by using kinetic partitioning and mass spectrometry with mutants of these back-door cysteines. Thus, the active-site cysteine is protected by rapid intramolecular disulfide formation with the back-door cysteines in the wild-type enzymes. These intramolecular disulfides can then be rapidly and effectively rereduced by thioredoxin/thioredoxin reductase but not glutathione. Thus, the chemistry and kinetics of the active-site cysteines of the Cdc25's support a physiological role for reversible redox-mediated regulation of the Cdc25's, important regulators of the eukaryotic cell cycle.

Original languageEnglish (US)
Pages (from-to)10060-10070
Number of pages11
JournalBiochemistry
Volume42
Issue number34
DOIs
StatePublished - Sep 2 2003
Externally publishedYes

ASJC Scopus subject areas

  • Biochemistry

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