We have studied the binding nature of an aromatic aldehyde to the catalytic site of liver alcohol dehydrogenase from horse (LADH) using preresonance Raman spectroscopy. the compound />-(dimethylamino)benzaldehyde (DA)is converted to the corresponding alcohol in the presence of nicotinamideadenine dinucleotide (NADH) and a catalytic amount of enzyme at neutral pH. A stable ternary complex of LADH/NADH/DABA can be formed if enzyme and coenzyme are in excessat high pH [Jagodzinski,P. W., Funk, G. F.,& Peticolas, W. L. (1982) Biochemistry 21, 2193-2202]. We have obtained the preresonance Raman spectrum of bound DABA by subtracting the contribution of the binary complex of LADH/NADH from thespectrum of this stable ternary complex. In order to understand the normal mode patterns of DABA, four isotopically labeled DABA derivatives were synthesized and their Raman spectra,in solution and in the ternary complex, were measured. Threeof these compounds contain substitutions in the functionally important aldehyde moiety: (i) In one such substitution, thealdehydic hydrogen atom was replaced by a deuterium; (ii) in another, this hydrogen atom was replaced by deuterium, and the aldehydic carbon atom was replaced by 13C; and (iii) in the third derivative, only the carbon atom was replaced by 13C. the fourth derivative has had the two hydrogen atoms at the 3- and 5-positions of the DABA ring replaced by deuterium atoms. We find that many of the spectral modes are fairly extended, involving both stretching and bending motions of the entire molecule, although a few modes are quite localized. We find that the normal mode structure of DABA changes considerably when it binds to LADH/NADH. As a model for the bound DABA, we have examined the zinc complexes of DABA (and all fourisotopically labeled samples) in anhydrous diethyl ether and methylene chloride. A striking correspondence between the Raman spectra of the enzyme-bound DABA and DABA-Zn complexes in solution is found, which extends to all the isotopically labeled derivatives. This suggests that one of the major roles of LADH in the binding of DABA is to provide a divalent zincion to form a first-sphere Lewis acid complex. the data also suggest other interactions between enzyme-bound DABA with its protein surroundings and with the coenzyme NADH are quite minor. An estimate of the carbonyl bond character of bound DABA had been made on the basis of the response of Raman bands to isotopic labeling and on trends observed in spectra of D BA in solvents of various polarities. It is found to have a bond order between a single and double bond. We discuss a possible role of the zinc ion at the enzyme's active site and subsequently the mechanism of the enzymatic function of LADH in view of these results.
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