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


Journal Article

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 NO2+) 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.

Full Text

Duke Authors

Cited Authors

  • Mohr, S; Stamler, JS; Brüne, B

Published Date

  • July 18, 1994

Published In

Volume / Issue

  • 348 / 3

Start / End Page

  • 223 - 227

PubMed ID

  • 8034046

Pubmed Central ID

  • 8034046

International Standard Serial Number (ISSN)

  • 0014-5793

Digital Object Identifier (DOI)

  • 10.1016/0014-5793(94)00596-6


  • eng

Conference Location

  • England