Inactivation of human glucose-6-phosphate dehydrogenase by peroxynitrite is modulated by bicarbonate and its catalytic ligands.

Abstract

Glucose-6-phosphate dehydrogenase, the rate-limiting enzyme of the pentose phosphate pathway, plays a key role in maintaining the redox status of cells. Here we report data on the oxidative inactivation of human glucose-6-phosphate dehydrogenase (hG6PDH) mediated by 10- or 40- fold molar excesses of peroxynitrite (ONOO-). Experiments were carried out in the absence or presence of bicarbonate (HCO3-), which is in equilibrium with CO2, and a recognized modulator of the ONOO- chemistry. Inactivation of hG6PDH (46.4 μM) by ONOO- was also examined in the presence of its substrates/co-factors, NADP+ and glucose 6-phosphate (G6P). Enzyme inactivation was quantified, mapping of modifications explored by LC-MS/MS and structural changes analyzed by circular dichroism, ANS fluorescence and SDS-PAGE. The data were rationalized by molecular dynamic simulations. hG6PDH was readily inactivated by ONOO-, with this mitigated by HCO3-, without significant changes in secondary structure. LC-MS/MS and peptide mass-mapping experiments provided evidence for oxidation at specific Met residues, and oxidation and nitration at Tyr and Trp. The Met sulfoxide yield was strongly modulated by HCO3-, while nitration of Tyr and Trp was minimally affected. NADP+ partially prevented hG6PDH inactivation, while an enhancement was observed for G6P. Molecular dynamics showed that the oxidized Met residues are located near a β-sheet close to the structural NADP+ binding site, potentially rationalizing the observed enzyme inactivation and NADP+-mediated protection. These results indicate that hG6PDH is highly susceptible to ONOO--mediated inactivation by mechanisms modulated by HCO3-, G6P, and NADP+, which can be relevant for future investigations at cellular level.

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