Vibrational analysis of NADH cofactors bound to glycerol-3-phosphate dehydrogenase and dogfish lactate dehydrogenase

The dehydrogenases catalyze the interconversions of aldehydes and alcohols in hydride transfer reactions to and from the re or si face of the nicotinamide ring of NAD with a high degree of stereospecificity. We report on the Raman spectra of NADH bound to dogfish lactate dehydrogenase (dfLDH), which transfers to and from the re face, and glycerol-3-phosphate dehydrogenase (G3PDH), which transfers to and from the si face. All theories of the mechanism of the stereospecificity must be in accord with the ground-state native protein-cofactor structure, and the vibrational spectrum of the dihydronicotinamide ring of NADH is sensitive to apoprotein-cofactor interactions. Our results show that the amide NH₂ rocking mode shifts up by about 30 cm^-1 when NADH is bound in dfLDH while virtually unchanged in G3PDH. The upward shift of the rocking mode indicates a stronger hydrogen bonding on the NH₂ moiety in dfLDH. Thus, the results are interpreted to show that the bound dihydronicotinamde head group interacts more strongly with surrounding protein and deforms to a greater extent in dfLDH than in G3PDH. It is concluded that a simple geometrical interpretation explaining the stereospecificity of hydride transfer in dehydrogenases is an adequate molecular description and that there is no need to invoke 'activation' of hydride transfer from one side of the NADH ring over the other. On the other hand, the results suggest that the fidelity of the stereospecificity for G3PDH should be much less than that found for LDH.