Rapid-scanning spectral evidence for catalytically nonequivalent but interconvertible forms of equine liver alcohol dehydrogenase.

This research investigated the reduction of p-nitrobenzaldehyde by NADH in equine liver alcohol dehydrogenase, indicating that the enzymatic reaction is biphasic and involves the oxidation of enzyme-bound reduced coenzyme. Importantly, the research revealed that the enzyme exists in two forms that can transform from one to another, and this process is facilitated by protomer interactions influenced by ligands.

Understanding the Nature of the Reaction

• In the kinetic studies of equine liver alcohol dehydrogenase-catalyzed reduction of p-nitrobenzaldehyde conducted under single turnover conditions, researchers discovered that the reaction is biphasic when NADD, a coenzyme, is used. The study further estimated the rate constants for the subsequent phases, the first phase occurring at around 200 sec-1 and the second at 0.5 sec-1. In addition, the amplitude ratio was found to be about 0.5, indicating that both phases are of equivalent magnitude.

Oxidation Process & Negligible Recycling

• Each phase of the reaction involves the oxidation of enzyme-bound reduced coenzyme. This means that during each phase, the enzyme is involved in breaking down the coenzyme through oxidation.
• The study found negligible recycling of the enzyme sites in the presence of 20 mM pyrazole. This indicates that pyrazole does not contribute significantly to the reutilization of these enzyme sites during the reaction process.

Interconversion of Enzyme Forms

• The research also showed that the rapid reaction rates involving the E(NAD-pyrazole) complex formation at every enzyme site and the oxidation of the coenzyme in the slower phase are limited by the same process.
• The researchers posited that the reaction’s biphasicity resulted from nonequivalent yet transformable forms of the enzyme operating. Significantly, the study suggests that the transformation or interconversion of these enzyme forms is driven by ligand-dependant protomer-protomer interactions. In this context, protomers are the individual subunits that compose a larger protein complex. Variations in these interactions, influenced by different ligands, allow the enzyme to exist in multiple functional states.