Resonance-enhanced Raman identification of a ternary chemical intermediate during the equine liver alcohol dehydrogenase reduction of p-(dimethylamino)benzaldehyde.

The nature of the binding of aromatic aldehyde and aromatic alcohol substrates to the catalytic zinc of equine liver alcohol dehydrogenase has been studied by using resonance-enhanced Raman spectroscopy. When an excess of both enzyme and coenzyme to substrate is used, a stable ternary chemical intermediate is formed between liver alcohol dehydrogenase and the reduced coenzyme, nicotinamide adenine dinucleotide, and the aldehyde, p-(dimethylamino)benzaldehyde, in the pH range 8.5-0.6. Resonance-enhanced Raman spectra clearly show that this same intermediate is formed between the excess enzyme, oxidized coenzyme, and the corresponding alcohol, p-(dimethylamino)benzyl alcohol. Thus, in the presence of excess enzyme and coenzyme, this specific ternary complex is a stable intermediate for both forward and reverse reactions. As a model for this enzyme-substrate intermediate, a complex between the aldehyde and Zn2+ in diethyl ether was made which showed a resonance-enhanced Raman spectrum essentially identical with that of the enzyme-coenzyme-substrate intermediate and completely different from that of the substrate. Most striking in this spectrum is the total absence of the carbonyl vibration which indicates that the C = O no longer exists in either the enzyme-substrate-coenzyme intermediate or the model complex, most probably due to the presence of a zinc-oxygen bond. The assignments are aided by 18O isotopic substitution in the substrate. The Raman spectra of crystals of the ternary complex and the dynamics of the complex are also discussed.