Active site titration of the tyrosine phosphatases SHP-1 and PTP1B using aromatic disulfides. Reaction with the essential cysteine residue in the active site.
Aromatic disulfides were found to inactivate truncated forms of the SHP-1 and PTP1B phosphatases by reaction with the essential active site cysteine residue. For truncated SHP-1 at pH 5.0, the reaction proceeded through an initial burst phase followed by a slower secondary phase. Our experiments demonstrated that the burst phase corresponded to the reaction of the aromatic disulfide with the active site cysteine. The magnitude of the burst phase was found to measure the active enzyme concentration, and the rate of the burst reflected the reactivity of the active site cysteine. The data were consistent with a mechanism in which an intramolecular disulfide is formed between the active site cysteine and a proximal cysteine during the burst reaction. Aromatic disulfides were found to react with the active site cysteines of full-length SHP-1 and truncated PTP1B also. Using vanadate to mask the active site cysteine, the active enzyme concentration could be assayed by comparing product yields for the reaction with aromatic disulfides in the presence and absence of vanadate at pH 8.0. These findings demonstrate the utility of aromatic disulfides as active site titrants and reactivity probes for tyrosine phosphatases.
Pregel
M J
MJ
Pharmaceutical Biotechnology Sector, Biotechnology Research Institute, National Research Council of Canada, Montréal, Québec H4P 2R2, Canada. mpregel@genzyme.com
Storer
A C
AC
D001665:Binding Sites; D003545:Cysteine / Q000378:metabolism*; D004220:Disulfides / Q000737:chemistry*; D004228:Dithionitrobenzoic Acid / Q000737:chemistry*; D047908:Intracellular Signaling Peptides and Proteins; D007700:Kinetics; D015335:Molecular Probes; D054592:Protein Tyrosine Phosphatase, Non-Receptor Type 11; D053824:Protein Tyrosine Phosphatase, Non-Receptor Type 6; D017027:Protein Tyrosine Phosphatases / Q000737:chemistry / Q000378:metabolism*
D004220:Disulfides; D047908:Intracellular Signaling Peptides and Proteins; D015335:Molecular Probes; D004228:Dithionitrobenzoic Acid; D054592:Protein Tyrosine Phosphatase, Non-Receptor Type 11; D053824:Protein Tyrosine Phosphatase, Non-Receptor Type 6; D017027:Protein Tyrosine Phosphatases; D003545:Cysteine