Catalytic and chemical competence of regulation of cdc25 phosphatase by oxidation/reduction.
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 active-site 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.
Sohn
Jungsan
J
Department of Biochemistry, Duke University Medical Center, Durham, North Carolina 27710, USA.
Rudolph
Johannes
J
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D002384:Catalysis; D020134:Catalytic Domain; D003545:Cysteine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000494:pharmacology; D005978:Glutathione / Q000737:chemistry / Q000378:metabolism; D006861:Hydrogen Peroxide / Q000737:chemistry / Q000494:pharmacology; D007527:Isoenzymes / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D008958:Models, Molecular; D010084:Oxidation-Reduction; D010710:Phosphates / Q000494:pharmacology; D021241:Spectrometry, Mass, Electrospray Ionization; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013434:Sulfenic Acids / Q000737:chemistry / Q000378:metabolism; D013880:Thioredoxin-Disulfide Reductase / Q000378:metabolism / Q000494:pharmacology; D013879:Thioredoxins / Q000494:pharmacology; D020687:cdc25 Phosphatases / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
D004791:Enzyme Inhibitors; D007527:Isoenzymes; D010710:Phosphates; D013434:Sulfenic Acids; D013879:Thioredoxins; D006861:Hydrogen Peroxide; D013880:Thioredoxin-Disulfide Reductase; D020687:cdc25 Phosphatases; D005978:Glutathione; D003545:Cysteine