Cysteine-sensitised formation and repair of mixed disulfide in the oxidation of papain by H2O2 and OH radicals

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Abstract
The inactivation of the proteolytic enzyme papain by H2O2 produces sulfenic acid by oxidation of the essential SH of cysteine 25 at the enzyme active site: .**GRAPHIC**. The kinetics of repair of this entity by cysteine were consistent with the 2 reactions: .**GRAPHIC**. Reaction 4 was the faster with k4 .gtoreq. 800 M-1 s-1, and k5 = 11.3 .+-. 0.5 M-1 s-1. A computer program was developed to evaluate the contributions of peroxide-inactivation and cysteine-repair when they occur simultaneously in N2O-saturated solutions in the absence of catalase. The yields predicted by this program agreed well with the inactivation caused by peroxide in irradiated systems. The effect of cysteine on the inactivation of papain by OH radicals produced by radiolysis of N2O-saturated solutions containing catalase was also investigated. Protection against permanent inactivation was much more efficient than expected on the basis of a simple competition between cysteine and papain for OH radicals, but there was a marked increase in the yield of repairable damage which was not due to H2O2. These observations can be qualitatively accounted for by the reactions: .**GRAPHIC**. The same rate constant was obtained for the repair of PapainCys25SSCys from this source as from the peroxide inactivation and treatment with cysteine. However, there was also evidence for additional cysteine-sensitized production of mixed disulfide and this probably occurs through reactions of CysS.cntdot. radicals: .**GRAPHIC**.

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