Mechanism of covalent modification of glyceraldehyde‐3‐phosphate dehydrogenase at its active site thiol by nitric oxide, peroxynitrite and related nitrosating agents

FEBS Letters

Stamler, Jonathan S.¹  Mohr, Susanne²  Brüne, Bernhard² 

[1] Duke University Medical Center, Pulmonary and Cardiovascular Divisions, PO Box 3177, Durham, NC 27710, USA;University of Konstanz, Faculty of Biology, PO Box 5560 M612, 78434 Konstanz, Germany

关键词: Nitric oxide;    Thiol modification;    GAPDH;    S-Nitrosylation;    GAPDH;    glyceraldehyde-3-phosphate dehydrogenase;    SNP;    sodium nitroprusside;    SIN-1;    3-morpholinosydnonimine;    DTT;    dithiothreitol;    NAD+;    nicotinamide adenine dinucleotide;   

DOI  :  10.1016/0014-5793(94)00596-6

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

Previous studies have suggested that glyceraldehyde-3-phosphate dehydrogenase (GAPDH) undergoes covalent modification of an active site thiol by a NO•-induced [³²P]NAD⁺-dependent mechanism. However, the efficacy of GAPDH modification induced by various NO donors was found to be independent of spontaneous rates of NO• release. To further test the validity of this mechanism, we studied the effects of nitrosonium tertrafluoroborate (BF₄NO), a strong NO⁺ donor. BF₄NO potently induces GAPDH labeling by the radioactive nucleotide. In this case, the addition of thiol significantly attenuates enzyme modification by competing for the NO moiety in the formation of RS—NO. Peroxynitrite (ONOO⁻) also induces GAPDH modification in the presence of thiol, consistent with the notion that this species can transfer NO⁺ (or NO⁺ ₂) through the intermediacy of RS—NO. However, the efficiency of this reaction is limited by ONOO⁻-induced oxidation of protein SH groups at the active site. ONOO⁻ generation appears to account for the modification of GAPDH by SIN-1. Thus, S-nitrosylation of the active site thiol is a prequisite for subsequent post-translational modification with NAD⁺, and emphasizes the role of NO⁺ transfer in the initial step of this pathway. Our findings thus provide a uniform mechanism by which nitric oxide and related NO donors initiate non-enzymatic ADP-ribosylation (like) reactions. In biological systems, endogenous RS—NO are likely to support the NO group transfer to thiol-containing proteins.

【 授权许可】

Unknown