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

M Xian; X Chen; Z Liu; K Wang; P G Wang

doi:10.1074/jbc.M001054200

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

Journal article

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

M Xian, X Chen, Z Liu, K Wang and P G Wang

The Journal of biological chemistry, Vol.275(27), pp.20467-20473

07/07/2000

DOI: https://doi.org/10.1074/jbc.M001054200

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https://hdl.handle.net/2376/109136

PMID: 10779505

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Abstract

Glutathione - analogs & derivatives

Enzyme Inhibitors - pharmacology

Spectrometry, Fluorescence

Enzyme Activation - drug effects

Glutathione - pharmacology

Nitroso Compounds - pharmacology

Sulfhydryl Compounds - metabolism

Disulfides - chemistry

Papain - antagonists & inhibitors

Mercaptoethanol

Cysteine - metabolism

Molecular Structure

Kinetics

S-Nitrosoglutathione

S-Nitrosothiols

Nitric Oxide - metabolism

Binding Sites

Fluorescent Dyes

Dithiothreitol - pharmacology

Papain - chemistry

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.

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Title: Inhibition of papain by S-nitrosothiols. Formation of mixed disulfides
Creators: M Xian - Department of Chemistry, Wayne State University, Detroit, Michigan 48202, USA
X Chen
Z Liu
K Wang
P G Wang
Publication Details: The Journal of biological chemistry, Vol.275(27), pp.20467-20473
Academic Unit: Chemistry, Department of
Publisher: United States
Grant note: GM 54074 / NIGMS NIH HHS
Identifiers: 99900547012301842
Language: English
Resource Type: Journal article