Mechanism of glyceraldehyde‐3‐phosphate dehydrogenase inactivation by tyrosine nitration

Abstract

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a multifaceted protein that is involved in numerous processes including glycolysis, translational silencing, transcriptional regulation of specific genes, and acting as a nitric oxide sensor. The precise mechanism on how GAPDH is targeted to these different roles is unclear but believed to involve specific posttranslational modification to the protein. Numerous studies have demonstrated that GAPDH is a target for tyrosine nitration. However, the site of modification and the molecular consequence have not been defined. Rabbit GAPDH with a reversibly protected catalytic cysteine was nitrated in vitro with tetranitromethane, resulting in complete loss of GAPDH catalytic activity. Nitration was estimated as 0.32 mol of nitrotyrosine residue per mole of GAPDH. Mass spectrometry analysis of nitrated GAPDH indicated that Tyr311 and Tyr317 were the sole sites of nitration. The X-ray crystal structure revealed that the distances between Tyr311 and Tyr317 and the cofactor nicotinamide adenine dinucleotide (NAD 1 ) were less than 7.2 and 3.7 Å , respectively, implying that nitration of these two residues may affect NAD 1 binding. This possibility was assessed using an NAD 1 binding assay, which showed that nitrated GAPDH was incapable of binding NAD 1 . Thus, these results strongly suggest that Tyr311 and Tyr317 nitration prohibits NAD 1 binding, leading to the loss of catalytic activity.

Selective Vulnerability of Synaptic Signaling and Metabolism to Nitrosative Stress

“…Presently, our understanding of the effects of tyrosine nitration on GAPDH activity is limited. However, one recent in vitro study demonstrated that the nitration of Tyr311 and Tyr317 inactivated purified rabbit GAPDH via a mechanism that involves the inhibition of NAD + binding; to exclude effects on thiol groups, these experiments were done with the reversible protection of thiols (206).…”

Expression of glyceraldehyde 3-phosphate dehydrogenase is enhanced in Leishmania spp naturally resistant to nitric oxide

“…Some studies have demonstrated that GAPDH is a target for tyrosine nitration (Palamalai and Miyagi, 2010;Bailey et al, 2011;Guingab-Cagmat et al, 2011). Therefore, any mechanism that reduces the susceptibility of the organism to nitrative stress might contribute to the development of NO resistance.…”

Tyrosine Modifications in Aging

“…Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a crucial protein for cellular metabolism and energy, is a common entry on proteomic lists of nitrated proteins, including studies of aging (17) and AD (43), likely due to its role as a sensor of NO (27). In a mechanistic study, Palamalai et al (27) recently determined that in vitro nitration of Tyr-311 and Tyr-317 in rabbit GAPDH by tetranitromethane resulted in loss of binding to NAD + , thereby destroying all catalytic activity of the enzyme. Computational analysis of the X-ray crystal structure predicted that these nitration sites are located very close to both the catalytic cysteine residue and the NAD + binding site ( Fig.…”