Inhibition of papain by S-nitrosothiols - Formation of mixed disulfides

S-Nitrosylation of protein thiols is one of the cellular regulatory mechani sms induced by NO. The cysteine protease papain has a critical thiol residu e (Cys(25)). It has been demonstrated that NO or NO donors such as sodium n itroprusside and N-nitrosoaniline derivatives can reversibly inhibit this e nzyme 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-p enicillamine-2, and the S-nitroso tripeptide acetyl-Phe-Gly-S-nitrosopenici llamine, exhibited different inhibitory activities toward the enzyme in a t ime- 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 en zyme with a fluorescent S-nitroso probe (S-nitroso-5-dimethylaminonaphthale ne-1-sulfonyl) resulted in the appearance of fluorescence of the protein, i ndicating the formation of a thiol adduct. Moreover, S-transnitrosylation i n 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.