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 8.5–9.6. 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.