Studies of coenzyme binding to rabbit muscle glyceraldehyde-3-phoshate dehydrogenase.
The binding of NAD+, NADH and adenosine diphosphoribose (Ado-PP-Rib) to a stable, highly active and nucleotide-free preparation of rabbit muscle glyceraldehyde-3-phosphate dehydrogenase (D-glyceraldehyde-3-phosphate: NAD+ oxidoreductase (phosphorylating), EC 1.2.1.12) has been studied. All three nucleotides quench the protein fluorescence to the same extent when they bind to the enzyme, and this property has been used to measure the dissociation constants for the two high-affinity binding sites for the nucleotides. The results indicate negative interactions between, or non-identify of, these two binding sites, to which NAD+ and NADH bind with similar affinity. The binding of NAD+ to the enzyme has been studied by spectrophotometric titrations at 360 nm. It appears that the binding of NAD+ to each of the four subunits of the enzyme contributes equally to the intensity of this 'Racker' band. The dissociation constants associated with the binding of the third and fourth molecules of NAD+ estimated from such titrations confirm some previous estimates. The binding of NADH to the enzyme causes a decrease of intensity of the absorbance of the coenzyme at 340 nm, and the dissociation constants for binding of the third and fourth molecules of NADH have been estimated from spectrophotometric titrations. They are the same as those for NAD+.  Judging by the apparent dissociation constants, negative interactions on binding the third molecule of NAD+ or NADH are more marked than those associated with the binding of the second and fourth molecules, suggesting that a major conformational change occurs at half-saturation of the tetramer with coenzyme.
D000247:Adenosine Diphosphate Sugars; D000818:Animals; D001051:Apoenzymes; D001665:Binding Sites; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000378:metabolism; D007700:Kinetics; D009132:Muscles / Q000201:enzymology*; D009243:NAD; D010084:Oxidation-Reduction; D011485:Protein Binding; D011817:Rabbits; D012266:Ribose; D013050:Spectrometry, Fluorescence; D013056:Spectrophotometry, Ultraviolet
D000247:Adenosine Diphosphate Sugars; D001051:Apoenzymes; D009243:NAD; D012266:Ribose; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases