The NAD(P) dependent dehydrogenases and reductases stereospecifically catalyze the transfer of a hydride ion from C4 of the dihydronicotinamide of the NAD(P) ring to the substrate. We have investigated the vibrational structure of the important C4−H coordinate for NAD(P)H and NADP+ bound to three enzymes in binary and ternary (Michaelis mimics) complexes:  the A-side specific lactate dehydrogenase (LDH) and dihydrofolate reductase (DHFR) and the B-side specific glycerol-3-phosphate dehydrogenase (G3PDH). This is achieved by specifically deuterating the C4 pro-R or pro-S hydrogens of the reduced ring or the C4 hydrogen of the oxidized ring, which results in a vibrational mode localized to the stretching motion of the labeled C4−H bond. We observed relatively minor changes in the stretch frequencies of the C4−H bonds showing that the electronic nature of the bond is not substantially modified by cofactor binding, a mechanism previously proposed to be involved in enzymic “activation” toward catalysis. However, from the observed band narrowing of the C4-D stretch band, it is clear that interactions at the active site in all three proteins greatly reduced the conformational flexibility of either the reduced or oxidized ring as the cofactor moves from solution to the binary complex or ternary complex, guiding the ring structure from the ensemble of structures accessible in solution toward a selected set. Moreover, as NAD(P)H binds to LDH or DHFR forming binary as well as ternary Michaelis mimic complexes, the pro-R hydrogen is brought to a pseudoaxial orientation, which is thought to be the proper geometry for the transition state of hydride transfer. Hence, ground state structural distortions imposed on the cofactor appear to populate preferentially the correct ring geometry for enzymic activity. Surprisingly, the mimics of their Michaelis complexes also contain a substantial second, presumably unproductive, population of the bound cofactor whereby the pro-S hydrogen is pseudoaxial. Unexpectedly, the geometry of NADH bound to G3PDH is nearly planar with the pro-R hydrogen slightly pseudoaxial. This would seem to be a poorly bound cofactor for catalysis although it may well be true that the transition state geometry for G3PDH is not that of LDH. How the results bear on various proposals concerning ground-state regulation of reactivity is discussed.

Continuum electrostatic calculations in conjunction with molecular dynamics simulations have been used to investigate the source of the stereospecificity in the hydride transfer reaction catalyzed by lactate dehydrogenase (LDH). These studies show that favorable electrostatic interactions between the carboxamide group of the reduced nicotinamide adenine dinucleotide coenzyme and protein residues of the active site of LDH can account for much if not all of the stereospecificity of the LDH-catalyzed reaction, with A-side hydride transfer more than 10(7) times greater than B-side transfer. Unfavorable steric interactions within the binding complex for B-side transfer are not found.

The isolated S-peptide (enzymatically cleaved 1-20 fragment of the ribonuclease, RNase S) is partly helical in solution. Both S-peptide and its truncated 1-15 analogue pep01 can combine with S-protein (residual part of RNase S without S-peptide), restoring a RNase S′ complex. Part 3-13 of the peptides forms an R-helix when within the RNase S′ complex. To compare the solvated forms of both peptides with their structures when complexed with the S-protein, we have measured the Raman spectra of these forms by means of difference spectroscopy. The spectra of the peptides in water solutions are characterized by much wider vibrational bands than the spectrum of the peptides within the RNase S′ complexes. This shows that the structure of the solvated peptides consists of a heterogeneous mixture of interconverting species. Only two main bands characteristic for the exposed R-helix (in D 2 O) and for an unordered chain are observed for amide-I regions of the dissolved peptides spectra. Relative intensities of the bands vary with temperature, reflecting different proportions of helical and disordered conformations. However, amide-I bands compositions of the bound peptides are more complex and include marker bands typical of the R-helix within the hydrophobic environment of a protein.