Inhibition of papain by S-Nitrosothiols: Formation of mixed disulfides

Abstract

S-Nitrosylation of protein thiols is one of the cellular regulatory mechanisms induced by NO. The cysteine protease papain has a critical thiol residue (Cys25). It has been demonstrated that NO or NO donors such as sodium nitroprusside and N-nitrosoaniline derivatives can reversibly inhibit this enzyme by S-NO bond formation in its active site. In this study, a different regulated mechanism of inactivation was reported using S- nitrosothiols as the NO donor. Five S-nitroso compounds, S-nitroso-N-acetyl- DL-penicillamine, S-nitrosoglutathione, S-nitrosocaptopril, glucose-S- nitroso-N-acetyl-DL-peniciliamine-2, and the S-nitroso tripeptide acetyl-Phe- Gly-S-nitrosopenicillamine, exhibited different inhibitory activities toward the enzyme in a time- and concentration-dependent manner with second-order rate constants (k(i)/K(I)) ranging from 8.9 to 17.2 M-1 S-1. The inhibition of papain by S-nitrosothiol was rapidly reversed by dithiothreitol, but not by ascorbate, which could reverse the inhibition of papain by NOBF4. Incubation of the enzyme with a fluorescent S-nitroso probe (S-nitroso-5-dimethylaminonaphthalene-1-sulfonyl) resulted in the appearance of fluorescence of the protein, indicating the formation of a thiol adduct. Moreover, S-transnitrosylation in the incubation of S-nitroso inactivators with papain was excluded. These results suggest that inactivation of papain by S-nitrosothiols is due to a direct attack of the highly reactive thiolate (Cys25) in the enzyme active site on the sulfur of S-nitrosothiols to form a mixed disulfide between the inactivator and papain.