Mechanism of glyceraldehyde-3-phosphate dehydrogenase inactivation by tyrosine nitration.
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(+)) were less than 7.2 and 3.7 A, respectively, implying that nitration of these two residues may affect NAD(+) binding. This possibility was assessed using an NAD(+) binding assay, which showed that nitrated GAPDH was incapable of binding NAD(+). Thus, these results strongly suggest that Tyr311 and Tyr317 nitration prohibits NAD(+) binding, leading to the loss of catalytic activity.
Palamalai
Vikram
V
Department of Biochemistry and Molecular Biology, University of North Dakota School of Medicine and Health Sciences, Grand Forks, North Dakota, USA.
Miyagi
Masaru
M
D000818:Animals; D055162:Biocatalysis; D018360:Crystallography, X-Ray; D004789:Enzyme Activation; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000737:chemistry* / Q000378:metabolism*; D008958:Models, Molecular; D009243:NAD / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011817:Rabbits; D014443:Tyrosine / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism
D009243:NAD; C002744:3-nitrotyrosine; D014443:Tyrosine; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases