Classical Raman spectroscopic studies of NADH and NAD+ bound to lactate dehydrogenase by difference techniques.
The binding of the coenzymes NAD+ and NADH to lactate dehydrogenase causes significant changes in the Raman spectra of both of these molecules relative to spectra obtained in the absence of enzyme. The molecular motions of the bound adenine moiety of both NAD+ and NADH as well as adenine containing analogues of these coenzymes produce Raman bands that are essentially identical, suggesting that the binding of adenine to the enzyme is the same regardless of the nicotinamide head-group nature. We also have observed that the molecular motions of the bound adenine moiety are different from both those obtained when it is in either water, various hydrophobic solvents, or various other solvent compositions. Protonation of the bound adenine ring at the 3-position is offered as a possible explanation. Significant shifts are observed in both the stretching frequency of the carboxamide carbonyl of NAD+ and the rocking motion of the carboxamide NH2 group of NADH. These shifts are probably caused by hydrogen bonding with the enzyme. The interaction energies of these hydrogen-bonding patterns are discussed. The aromatic nature of the nicotinamide moiety of NAD+ appears to be unchanged upon binding. Pronounced changes in the Raman spectrum of the nicotinamide moiety of NADH are observed upon binding; some of these changes are understood and discussed. Finally, these results are compared to analogous results that were recently reported for liver alcohol dehydrogenase [Chen et al. (1987) Biochemistry 26, 4776-4784]. In general, the coenzyme binding properties are found to be quite similar, but not identical, for the two enzymes.
Deng
H
H
Physics Department, City College of City University of New York, New York 10031.
Zheng
J
J
Sloan
D
D
Burgner
J
J
Callender
R
R
D000818:Animals; D006860:Hydrogen Bonding; D007770:L-Lactate Dehydrogenase / Q000378:metabolism*; D009243:NAD / Q000378:metabolism*; D010084:Oxidation-Reduction; D011485:Protein Binding; D011487:Protein Conformation; D012997:Solvents; D013059:Spectrum Analysis, Raman / Q000379:methods; D013552:Swine
D012997:Solvents; D009243:NAD; D007770:L-Lactate Dehydrogenase