The sequential nature of the negative cooperativity in rabbit muscle glyceraldehyde-3-phosphate dehydrogenase

The binding of nicotinamide–adenine dinucleotide (NAD+), nicotinamide‐1, N6‐ethenoadenine dinucleotide (ɛNAD+), acetylpyridine–adenine dinucleotide (AcPyAD+), ATP, and adenosine diphosphoribose (ADP‐ribose) to rabbit muscle glyceraldehyde‐3‐phosphate dehydrogenase (the enzyme) was examined. NAD+ and ɛNAD+ were found to bind to the apoenzyme in a negatively cooperative manner, whereas AcPyAD+, ATP, and ADP‐ribose bind non‐cooperatively to the NAD+ sites. The strong negative cooperativity in coenzyme binding was found to be abolished in the presence of AcPyAD+ and strongly weakened by ATP, ADP, and AMP, but was not affected by the addition of ADP‐ribose. These findings demonstrate that the mechanism of the negative cooperativity in coenzyme binding to the enzyme involves ligand‐induced conformational changes between neighboring sites. These findings cannot be accounted for by the pre‐existent asymmetry model. The results support our previous hypothesis that the structure of the pyridine moiety of the coenzyme analogues plays a role in orienting the adenine moiety in the adenine subsite, and thus affects the cooperativity observed in the binding of the coenzyme analogue.