ACTIVE-SITE TITRATION OF THE TYROSINE PHOSPHATASES SHP-1 AND PTP1B USING AROMATIC DISULFIDES - REACTION WITH THE ESSENTIAL CYSTEINE RESIDUEIN THE ACTIVE-SITE

Aromatic disulfides were found to inactivate truncated forms of the SH P-1 and PTP1B phosphatases by reaction with the essential active site cysteine residue. For truncated SHP-1 at pH 5.0, the reaction proceede d 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 c oncentration, and the rate of the burst reflected the reactivity of th e active site cysteine. The data were consistent with a mechanism in w hich an intramolecular disulfide is formed between the active site cys teine and a proximal cysteine during the burst reaction. Aromatic disu lfides were found to react with the active site cysteines of full-leng th SHP-1 and truncated PTP1B also. Using vanadate to mask the active s ite cysteine, the active enzyme concentration could be assayed by comp aring 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 reacti vity probes or tyrosine phosphatases.