Inhibition of papain by S-nitrosothiols. Formation of mixed disulfides.
S-Nitrosylation of protein thiols is one of the cellular regulatory mechanisms induced by NO. The cysteine protease papain has a critical thiol residue (Cys(25)). 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-penicillamine-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 NOBF(4). 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 (Cys(25)) in the enzyme active site on the sulfur of S-nitrosothiols to form a mixed disulfide between the inactivator and papain.
Xian
M
M
Department of Chemistry, Wayne State University, Detroit, Michigan 48202, USA.
Chen
X
X
Liu
Z
Z
Wang
K
K
Wang
P G
PG
D001665:Binding Sites; D003545:Cysteine / Q000378:metabolism; D004220:Disulfides / Q000737:chemistry; D004229:Dithiothreitol / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D005456:Fluorescent Dyes; D005978:Glutathione / Q000031:analogs & derivatives / Q000494:pharmacology; D007700:Kinetics; D008623:Mercaptoethanol; D015394:Molecular Structure; D009569:Nitric Oxide / Q000378:metabolism; D009603:Nitroso Compounds / Q000494:pharmacology*; D010206:Papain / Q000037:antagonists & inhibitors* / Q000737:chemistry; D026422:S-Nitrosoglutathione; D026403:S-Nitrosothiols; D013050:Spectrometry, Fluorescence; D013438:Sulfhydryl Compounds / Q000378:metabolism
D004220:Disulfides; D004791:Enzyme Inhibitors; D005456:Fluorescent Dyes; D009603:Nitroso Compounds; D026403:S-Nitrosothiols; D013438:Sulfhydryl Compounds; D009569:Nitric Oxide; D026422:S-Nitrosoglutathione; D008623:Mercaptoethanol; C032635:S-nitrosomercaptoethanol; D010206:Papain; D005978:Glutathione; D003545:Cysteine; D004229:Dithiothreitol