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Mechanism of covalent modification of glyceraldehyde‐3‐phosphate dehydrogenase at its active site thiol by nitric oxide, peroxynitrite and related nitrosating agents
Author(s) -
Mohr Susanne,
Stamler Jonathan S.,
Brüne Bernhard
Publication year - 1994
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/0014-5793(94)00596-6
Subject(s) - peroxynitrite , chemistry , thiol , covalent bond , glyceraldehyde 3 phosphate dehydrogenase , nitric oxide , active site , biochemistry , dehydrogenase , enzyme , organic chemistry , superoxide
Previous studies have suggested that glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH) undergoes covalent modification of an active site thiol by a NO•‐induced [ 32 P]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 (BF 4 NO), a strong NO + donor. BF 4 NO 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.