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

The research examines how aromatic aldehyde and aromatic alcohol substrates interact with the zinc catalyst found in equine liver alcohol dehydrogenase. By using resonance-enhanced Raman spectroscopy, a stable ternary chemical intermediate was observed during the enzyme’s reaction process involving coenzyme nicotinamide adenine dinucleotide and aldehyde p-(dimethylamino)benzaldehyde.

Study on Enzyme-Substrate Interactions

• The research investigated the binding properties of aromatic aldehyde and aromatic alcohol substrates with the zinc catalyst in equine liver alcohol dehydrogenase.
• Resonance-enhanced Raman spectroscopy was used to analyze this interaction, which is a technique that provides information about the vibrational modes of a system, allowing for analysis of molecular structure and interactions.

Formation of Ternary Chemical Intermediate

• Results showed that when both the enzyme and coenzyme were in excess with respect to the substrate, a stable intermediate compound formed between the liver alcohol dehydrogenase, the reduced coenzyme known as nicotinamide adenine dinucleotide, and the aldehyde, p-(dimethylamino)benzaldehyde.
• This intermediate formation was consistent in the pH range from 8.5 to 0.6.
• The Raman spectra also showed that this intermediate compound also formed in the presence of excess enzyme and corresponding alcohol p-(dimethylamino)benzyl alcohol and oxidized coenzyme, indicating that the intermediate is stable for both forward and reverse reactions.

Model and Observations

• A model intended to replicate the conditions for this enzyme-substrate intermediate was created. The model comprised of a complex between the aldehyde and Zn2+ in diethyl ether and exhibited a resonance-enhanced Raman spectrum similar to that of the enzyme-coenzyme-substrate intermediate.
• Remarkably, the carbonyl vibration was completely absent in this spectrum, suggesting that the carbonyl group C = O no longer exists in the enzyme-substrate-coenzyme intermediate or the model complex. This was likely the result of a bond formation between zinc and oxygen.
• These findings were supported by 18O isotopic substitution in the substrate, which helped in confirming the assignments.

Raman Spectra and Dynamics Analysis

• Finally, the researchers discussed the Raman spectra of crystals of the ternary complex and the dynamics of the complex formation, demonstrating further evidence for the observations made in the study.