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

Abstract

Chenl. 54, 242 (1976).The inactivation of the proteolytic enzyme papain by hydrogen peroxide produces a sulfenic acid by oxidation of the essential SH of cysteine 25 at the enzyme active site:The kinetics of repair of this entity by cysteine were consistent with the two reactions:[4 1 papainCys25SOH + CysSH 4 papainCys2sSSCys + H20 151 papainCys2sSSCys + CysSH -r papainCys2sSH + CysSSCys Reaction 4 was the faster with k4 2 800 M-1 s-1, and ks = 11.3 T 0.5 M-1 s-1. A computer program was developed to evaluate the contributions of peroxide-inactivation and cysteinerepair when they occur sinlultaneously in NzO-saturated solutions in the absence of catalase. The yields predicted by this program agreed well with the inactivation caused by peroxide in irradiated systelns.The effect of cysteine 011 the inactivation of papain by O H radicals produced by radiolysis of NlO-saturated solutions containing catalase was also investigated. Protection against permanent inactivation was n~uch more efficient than expected on the basis of a simple competition between cysteine and papain for O H radicals, but there was a marked increase in the yield of repairable damage which was not due to hydrogen peroxide. These observations can be qualitatively accounted for by the reactions:The same rate constant was obtained for the repair of PapainCys2jSSCys 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 t h r o~~g h reactions of CysS. radicals: Chem. 54, 242 (1976). L'inactivation de I'enzyme protkolytique de la papaine par le peroxyde d'hydrogkne conduit h la formation d '~~n acide sulfknique par oxydation du SH essentiel de la cystiine 25 au niveau du site actif de I'enzyme: O n a aussi etudii I'effet de la cystiine sur I'inactivatio~l de la papaine par les r a d i c a~~x O H p r o d~~i t s par la radiolyse de solutions saturies de N?O contenant la catalase. La protection contre une inactivation permanente est beaucoup plus eficace qu'attendue sur la base d'une simple conlpititio~l entre la cysteine et la papaine pour les radicaux O H , ~n a i s il y avait une augmentation marquee d a m le rendenlent de dornmage reparable qui n'itait pas dfi au peroxyde d'liydrogene. O n peut tenir compte qualitativenle~lt de ces observatiolls par les riactions:p a p a i n e C y~~~S .-+ (donne des pertes de S H non-riparables)On a obtenu les mimes constantes de vitesse pour la riparation de la papaineCys25SSCys B partir de cette source et aussi B partir d'une inactivation par le peroxyde et le traitenlent avec la cysteine. Toutefois, il y a aussi des indiquations pour la production se~lsibilisie par la cysteine du disulfure rnixte et ceci se produit vraisemblablenle~lt par des reactions des radicaux CysS.: