8-Alkylaminoadenyl nucleotides as probes of dehydrogenase interactions with nucleotide analogs of different glycosyl conformation

The nucleotides 8-amino-, 8-methylamino-, and 8-dimethylaminoadenylic acid have been synthesized and their preferred conformations about the glycosyl bond in qaueous solution have been determined by 1H nuclear magnetic resonance spectroscopy. Paramagnetic relaxation studies, nuclear Overhauser enhancement measurements, chemical shifts, and coupling constant comparisons indicate that their is rotation about the glycosyl bond and that preference for either the anti or syn conformation depends on the extent of alkyl substitution on the 8-amino group. The primary and secondary amines 8-amino- and 8-methylaminoadenylic acid adopt a perferential anti conformation about the glycosyl bond, while the tertiary amine 8-dimethylaminoadenylic acid exists predominantly in the syn form. These three analogs provide a system to study interactions of a dehydrogenase with coenzyme inhibitors having different glycosyl conformer populations. All three analogs are competitive inhibitors of NADH in reaction with chicken muscle lactate dehydrogenase, and the Ki values show little dependence on the nature of the amino substitution. This demonstrates that the distribution of conformations about the nucleotide glycosyl bond does not effect the competition of the nucleotide for lactate dehydrogenase apoenzyme. Several models for enzyme-coenzyme binding are discussed. The available data cannot distinguish whether the enzyme binds nucleotide in both the anti and syn conformations or in purely the anti conformation. However, at some stage of the enzyme-coenzyme interaction, there appears to be a strong stabilization of the nucleotide in the anti conformation about the glycosyl bond.