Raman spectroscopic studies of NAD coenzymes bound to malate dehydrogenases by difference techniques.
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.(ABSTRACT TRUNCATED AT 250 WORDS)
Deng
H
H
Physics Department, City College of the City University of New York 10031.
Burgner
J
J
Callender
R
R
D003067:Coenzymes / Q000737:chemistry*; D003593:Cytoplasm / Q000737:chemistry / Q000201:enzymology; D007770:L-Lactate Dehydrogenase / Q000737:chemistry; D008291:Malate Dehydrogenase / Q000737:chemistry*; D008928:Mitochondria / Q000737:chemistry / Q000201:enzymology; D009243:NAD / Q000737:chemistry* / Q000031:analogs & derivatives; D011485:Protein Binding; D013059:Spectrum Analysis, Raman
D003067:Coenzymes; D009243:NAD; C005941:3-acetylpyridine adenine dinucleotide; D007770:L-Lactate Dehydrogenase; D008291:Malate Dehydrogenase