title: The nicotinamide subsite of glyceraldehyde-3-phosphate dehydrogenase studied by site-directed mutagenesis.
abstract: Directed mutagenesis has been used to study the nicotinamide subsite of the glycolytic NAD-dependent glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Residue Asn313 is involved together with the carboxyamide moiety of the nicotinamide ring in a complex network of hydrogen bonding interactions which fix the position of the pyridinium ring of NAD to which hydride transfer occurs at the C-4 position in the catalytic reaction. The asparagine side-chain has been replaced by that of the Thr and Ala residues and results in mutants with very similar properties. Both mutants show much weaker binding of NAD and lower catalytic efficiency. The mutant Asn313----Thr still exhibits strict B-stereospecificity in hydride transfer and retains the property of negative co-operativity in NAD binding. These experiments strongly suggest that the mutant enzyme undergoes the apo----holo sub-unit structural transition associated with coenzyme binding but that the nicotinamide ring is no longer as rigidly held in its pocket as in the wild type enzyme. The results shed light on the details of the molecular interactions which are responsible for negative co-operativity in this enzyme.
authors: URA CNRS 457, Faculté des Sciences, Vandoeuvre-lès-Nancy, France.
authors: C
authors: C
authors: Corbier
authors: A
authors: A
authors: Mougin
authors: Y
authors: Y
authors: Mely
authors: H W
authors: HW
authors: Adolph
authors: M
authors: M
authors: Zeppezauer
authors: D
authors: D
authors: Gerard
authors: A
authors: A
authors: Wonacott
authors: G
authors: G
authors: Branlant
mesh_terms: D001051:Apoenzymes / Q000737:chemistry / Q000235:genetics; D003903:Deuterium; D004789:Enzyme Activation; D001411:Geobacillus stearothermophilus / Q000201:enzymology* / Q000235:genetics; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000737:chemistry* / Q000235:genetics; D006358:Hot Temperature; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D009243:NAD / Q000737:chemistry* / Q000378:metabolism; D010766:Phosphorylation; D011487:Protein Conformation; D013237:Stereoisomerism; D014961:X-Ray Diffraction
chemical_list: D001051:Apoenzymes; D009243:NAD; D003903:Deuterium; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases