effects of NAD+ binding on the luminescence of Trp84 and Trp310. NAD+-induced conformational change is sequential and subtle rearrangement in the structure of unligated subunits might be responsible for the negative cooperative behavior of NAD+ binding
activity with D-glyceraldehyde 3-phosphate in presence of 0.0006-0.006 mM NAD+: activity of the monomeric form is negligible or absent, high activity of the tetramer. 0.02-0.06 mM NAD+: markedly higher catalytic activity of the monomer. 2 mM NAD+: specific activities of monomeric and tetrameric forms are similar and equal to 0.06 mM/min. Activity with 3-phospho-D-glyceroyl phosphate: activity of the monomer is considerably higher than that of the tetramer
1 NAD+ molecule per subunit, all four subunits have NAD+ bound in the active site, enzyme binding structure of wild-type and mutant C152S enzymes, overview
binding structure analysis: the NAD+ molecule inserts its pyrimidine ring into a narrow hydrophobic binding pocket on the positively charged surface of GAPDH3. The residues Asn7, Gly8, Phe9, Gly10, Asp33, Pro34, Phe35, The97, Gly98, and Phe100 tightly enclose the pyrimidine ring of NAD+. Because of a common adenine head, adenosine can also bind tightly to this hydrophobic binding pocket. There are two consecutive positively charged regions that contain an NAD+-binding site, one of which is located on each side of the tetramer
differing occupancy by NAD, bGAPDH(NAD)4, and bGAPDH(NAD)3 in the homotetramer, structure analysis, overview. Importance of Phe34 in NAD+ binding, Phe34 is stabilized in the presence of NAD+ but displays greater mobility in its absence. The oxidative state of the active site Cys149 residue is regulated by NAD+ binding, because this residue is found oxidized in the absence of dinucleotide. The distance between Cys149 and His176 decreases upon NAD binding and Cys149 remains in a reduced state when NAD+ is bound. Dual side-chain conformations are observed in Ser207 in subunits of O, Q, and R of the bGAPDH(NAD)3. Residues Pro33 and Phe34 form a bottleneck for NAD+ binding
monomer interactions with all three neighbor subunits are essential for maintaining the cooperativity of NAD+ binding exhibited by GAPD, NAD+ binding analysis, kinetics, overview
NAD+ binding analysis with truncated and mutant enzymes, kinetics, overview. The enzyme exhibits positive cooperativity with the saturation being reached at the NAD+/protein concentration ratio of about 20. The E96Q and E244Q substitutions do not alter the NAD+-binding behavior of dN-GAPDS significantly. The mutant proteins exhibit a well-pronounced positive cooperativity in coenzyme binding, while mutant dN-GAPDS D311N binds NAD+ noncooperatively
analysis of the structure of the cofactor-enzyme complex (PDB ID 6PX2) reveals variable NAD+ occupancy across the four monomers of the tetrameric enzyme. The NAD+ binding sites of the tetramer are occupied to different extents, overview
binding site and binding structure, detailed overview. The NAD+ binding domain consist of six parallel and one anti-parallel beta-strands, surrounded by four alpha-helices, the typical structure of a Rossmann fold
enzyme binding structure analysis. Subunit A contains a NAD+ molecule and a sulfate ion in the holo state. The NAD-binding site located in the N-terminal domain. The O atom of the nicotinamide ribose of NAD is hydrogen-bonded to the hydroxyl group in the side chain of Thr121. The NAD+ diphosphate moiety binds to the main chain of the protein with hydrogen bonds from the backbone amino groups of Arg12 and Ile13. The two hydroxyl groups of the adenosine ribose of the NAD+ are hydrogen-bonded to Asp34, and Arg78 forms a hydrogen bond to the amide group of the adenine base
wild-type enzyme has no activity with NADP+. The mutant enzyme D32A/L187N shows catalytic efficiency with NADP+ higher than that with NAD+. Activity of mutant enzyme D32A with NAD+ is equal to that of the wild-type enzyme. Activity of mutant L187N with NAD+ is higher than that of the wild-type enzyme. Mutant enzymes D32A and L187N also show activity with NADP+, 3-7% of the activity with NAD+
the coenzyme analogue binds in a non-productive manner, resulting in a disordered thionicotinamide ring and rearranged active-site residues, effective as substrate to replace NAD+
the wild-type enzyme shows no activity with NADP+. The carboxylate group of Asp35 forms a network of hydrogen bonds with the 2' and 3-hydroxylgroups of the adenosine ribose ring of NAD+. This seems to be a critical factor to discriminate against the 2'-phosphate group of NADP+, because of electrostatic repulsion between the carboxylate group and phosphate group. Mutation of residues D35, L36, T37, and P192 alters the enzyme's cofactor specificity. Molecular docking and modelling
the wild-type enzyme shows no activity with NADP+. The carboxylate group of Asp35 forms a network of hydrogen bonds with the 2' and 3-hydroxylgroups of the adenosine ribose ring of NAD+. This seems to be a critical factor to discriminate against the 2'-phosphate group of NADP+, because of electrostatic repulsion between the carboxylate group and phosphate group. Mutation of residues D35, L36, T37, and P192 alters the enzyme's cofactor specificity. Molecular docking and modelling