A Raman spectroscopic characterization of bonding in the complex of horse liver alcohol dehydrogenase with NADH and N-cyclohexylformamide.
The binding of N-cyclohexylformamide (CXF) to the complex of horse liver alcohol dehydrogenase with NADH mimics that of the Michaelis complex for aldehyde reduction catalyzed by the enzyme. The Raman spectra of bound CXF and its 13C- and 15N-substituted derivatives have been obtained using Raman difference techniques, and the results are compared with CXF spectra in aqueous solution and in methylene chloride. The results indicate that the amide N-H bond is trans to the C=O bond of CXF both in solution and in the enzyme ternary complex. The C=O stretch and N-H bending modes of the amide of CXF shift -16 and -9 cm-1, respectively, in the enzyme ternary complex relative to that in aqueous solution and -48 and 36 cm-1, respectively, relative to that in methylene chloride. Ab initio normal mode calculations on various model systems of CXF show that the observed frequency changes of the C=O stretch mode have contributions from the frequency changes induced by the environmental changes near both the local C=O bond and the remote N-H bond. The same is true for the observed N-H bending frequency change. Our calculations also show that the environmentally induced frequency changes are additive so that it is possible to determine the C=O stretch (or N-H bending) frequency change that is due to the local interaction change near the C=O (or N-H) bond from the observed frequency changes. On the basis of these results and the empirical relationship between the C=O stretch frequency shift and the interaction enthalpy change on the C=O bond developed here, it is found that the C=O group of CXF in the enzyme/NADH/CXF complex binds with a favorable interaction enthalpy of approximately 5.5 kcal/mol relative to water. Similar analysis suggests that the N-H moiety of CXF is destabilized in the ternary complex by about 1.5 kcal/mol relative to water but is stabilized by about 1.5 kcal/mol relative to a hydrophobic environment. The analysis describes quantitatively the binding of the C=O of CXF with the catalytic zinc and the hydroxyl group of Ser-48 and the interaction of the N-H with the benzene ring of Phe-93 of the enzyme.
Department of Biochemistry, Albert Einstein College of Medicine, Bronx, New York 10461, USA.
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H
Deng
J F
JF
Schindler
K B
KB
Berst
B V
BV
Plapp
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R
Callender
D000426:Alcohol Dehydrogenase / Q000037:antagonists & inhibitors / Q000737:chemistry*; D000818:Animals; D005559:Formamides / Q000737:chemistry*; D006736:Horses; D006860:Hydrogen Bonding; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D046911:Macromolecular Substances; D009682:Magnetic Resonance Spectroscopy; D008432:Mathematical Computing; D008956:Models, Chemical; D009243:NAD / Q000737:chemistry*; D011485:Protein Binding; D013059:Spectrum Analysis, Raman / Q000379:methods; D013816:Thermodynamics
D005559:Formamides; D046911:Macromolecular Substances; D009243:NAD; C105265:N-cyclohexylformamide; D000426:Alcohol Dehydrogenase