Redox regulation of a soybean tyrosine-specific protein phosphatase.
Plant protein tyrosine phosphatases (PTPs) are important in regulating cellular responses to redox change through their reversible inactivation under oxidative conditions. Studies on the soybean (Glycine max) GmPTP have shown that, compared with its mammalian counterparts, the plant enzyme is relatively insensitive to inactivation by H2O2 but hypersensitive (k(inact) = 559 M(-1) s(-1)) to S-glutathionylation (thiolation) promoted by the presence of oxidized glutathione (GSSG). Through a combination of chemical and mutational modification studies, three of the seven cysteine residues of GmPTP have been identified by mass spectrometry as being able to inactivate the enzyme when thiolated by GSSG or alkylated with iodoacetamide. Conserved Cys 266 was shown to be essential for catalysis but surprisingly resistant to S-modification, whereas the regulatory Cys 78 and Cys 176 were readily thiolated and/or alkylated. Mutagenesis of these cysteines showed that all three residues were in proximity of each other, regulating each's reactivity to S-modifying agents. Through a combination of protein modification and kinetic experiments, we conclude that the inactivation of GmPTP by GSSG is regulated at two levels. Cys 176 appears to be required to promote the formation of the reduced form of Cys 266, which is otherwise unreactive. When thiolated, Cys 176 immediately inactivates the enzyme, and this is followed by the thiolation of Cys 78, which undergoes a slow disulfide exchange with Cys 266 giving rise to a Cys 78-Cys 266 disulfide. We speculate that this two-tiered protection is required for regulation of GmPTP under highly oxidizing conditions.
Dixon
David P
DP
School of Biological and Biomedical Sciences, University of Durham, South Road, Durham DH1 3LE, UK.
Fordham-Skelton
Anthony P
AP
Edwards
Robert
R
D000595:Amino Acid Sequence; D001665:Binding Sites / Q000235:genetics; D003545:Cysteine / Q000737:chemistry / Q000235:genetics; D004220:Disulfides / Q000737:chemistry / Q000378:metabolism; D005978:Glutathione / Q000737:chemistry / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction; D010940:Plant Proteins / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism*; D017027:Protein Tyrosine Phosphatases / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism; D012694:Serine / Q000235:genetics; D013025:Glycine max / Q000201:enzymology* / Q000235:genetics
D004220:Disulfides; D010940:Plant Proteins; D011994:Recombinant Proteins; D012694:Serine; D017027:Protein Tyrosine Phosphatases; D005978:Glutathione; D003545:Cysteine