We report here on the Raman spectra of NADH, 3-acetylpyridine adenine dinucleotide, APAD+, and a fragment of these molecules, adenosine 5′-diphosphate ribose (ADPR) bound to the mitochondrial (mMDH) and cytoplasmic (or soluble, sMDH) forms of malate dehydrogenase. We observe changes in the Raman spectrum of the adenosine moiety of these cofactors upon binding to mMDH, indicating that the binding site is hydrophobic. On the other hand, there is little change in the spectrum of the adenosine moiety when it binds to sMDH. Such observations are in clear contrast with those results obtained in LDH and LADH, where there are significant changes in the spectrum of the adenosine moiety when it binds to these two proteins. A strong hydrogen bond is postulated to exist between amide carbonyl group of NAD+ and the enzyme in the binary complexes with both mMDH and sMDH on the basis of a sizable decrease in the frequency of the carbonyl double bond. The interaction energy for formation of a hydrogen bond is the same as found previously for LDH, and we estimate that it is 2.8 kcal/mol more favorable in the binary complex than in water. A hydrogen bond is also detected between the amide -NH2 group of NADH and sMDH that is stronger than that formed in water and is of the same size as found in LDH. Surprisingly, the hydrogen bond to the -NH2 group in mMDH is the same as that found for water. The strength of these hydrogen bonds likely contributes to the varying observed shifts in the near-UV absorbance of the reduced nicotinamide when it binds to these enzymes. The lack of increased hydrogen-bond strength in the binary complex of NADH with mMDH relative to that found in water is proposed as a partial explanation of the red-shift in λmax found for reduced binary complex with mMDH, which is unusual for an A-side dehydrogenase. On the basis of the strength of the observed hydrogen-bond interactions between the acetyl moiety of APAD+ and the enzyme in the binary complex, we estimate that the energy difference between the syn and anti conformers of the pyridinium ring of the bound cofactor is at least 7.3 kcal/mol in mMDH and substantially more than this in sMDH and LDH where stronger interactions with the amide -NH2 can occur. This energy difference is sufficient, or nearly so, to determine the high degree of stereospecificity that is observed with these enzymes during transfer the pro-R hydrogen from NADH to substrate (LaReau et al., 1989).
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