THE CONFORMATION OF NAD(-DEHYDROGENASE DETERMINED BY NUCLEAR-MAGNETIC-RESONANCE WITH SUPPRESSION OF SPIN-DIFFUSION() BOUND TO LACTATE)

We have reinvestigated the conformation of NAD(+) bound to dogfish lac tate dehydrogenase (LDH) by using an NMR experiment that allows one to exploit nuclear Overhauser effects to determine internuclear distance s between pairs of protons, without perturbation of spin-diffusion eff ects from other protons belonging either to the cofactor or to the bin ding pocket of the enzyme. The analysis indicates that the structure o f bound NAD(+) is in accord with the conformation determined in the so lid state by x-ray diffraction for the adenosine moiety, but deviates significantly from that of the nicotinamide. The NMR data indicate con formational averaging about the glycosidic bond of the nicotinamide nu cleotide. In view of the strict stereospecificity of catalysis by LDH and the conformational averaging of bound NAD(+) that we infer from so lution-state NMR, we suggest that LDH binds the cofactor in both syn a nd anti conformations, but that binding interactions in the syn confor mation are not catalytically productive.