Structural and mechanistic aspects of alcohol dehydrogenase function

Abstract

Vertebrates possess a complex alcohol dehydrogenase (ADH) system composed of multiple molecular forms, which are currently classified into seven classes according to their structural properties. ADHs are dimeric zinc metalloenzymes that catalyze the reversible oxidation of alcohols to aldehydes/ketones using NAD+/NADH as electron acceptor and donor, respectively. The classes have broad but only partially overlapping substrate repertoires. This thesis mainly deals with mechanistic aspects of ADH function. The main focus is on the catalytic properties and possible physiological functions of ADH2. Structural studies of the native enzyme and of mutated forms establish the "functional window" in which ADH2 operates, and give in addition some general insights into dehydrogenase catalysis.

The tissue distribution of the five known human classes of ADH was determined by assessing rnRNA expression in 23 adult and four fetal tissues. The expression patterns were distinctly different. ADH1 and ADH3 were widely distributed while ADH2 and ADH5 were concentrated in the liver and ADH4 was concentrated in the stomach. ADH5 was predominantly found as a fetal form in the liver. These results suggest that there are several possible locations for ethanol metabolism in addition to the liver.

Human ADH1 and ADH2, but not ADH3, function as aldehyde dehydrogenases. The reaction proceeds by a ping-pong mechanism with aldehyde being both oxidized and reduced, which results in equimolar concentrations of the two products, alcohol and carboxylic acid. ADH1C was the most efficient isozyme in aldehyde oxidation. As a general rule, alcohols are preferred substrates even for the ADH1C-NAD+ complex, although in some cases aldehydes can be equally good. This is shown by experiments with the serotonin metabolite 5-hydroxy-indole-3-acetaldehyde.

ADH2 variants from mouse and rabbit were cloned and characterized together with the previously cloned variants from human and rat. Phylogenetic analysis showed that ADH2 is a remarkable divergent enzyme, although rat and mouse ADH2 were unexpectedly similar. These two rodent ADH2s had several unique properties, and, most importantly, their catalytic efficiency for various alcohols was two to three orders of magnitude lower than other ADHs. In rabbit liver, two isoforms of ADH2 were found, one of which was a low activity variant with no activity for long aliphatic alcohols. The variation in activity profile is not compatible with a general metabolic function, but rather implies that detoxification mechanisms are species-specific.

The mouse ADH2 structure has a novel topology of the substrate-binding pocket, which provides the structural basis for understanding the unique substrate and inhibitor repertoire of this enzyme. The two subunits of the dimer have different semi-opened interdomain conformations. Both conformations resemble that of ADH3, and are half way between the opened apo- and the closed holo-complex of ADH1. The activity of the mouse ADH2 was restored by replacing proline 47 with histidine. The structure of this high activity mutant emphasizes the importance of a short hydride transfer distance for efficient catalysis, and provides further evidence that formation of an alcoholate intermediate is crucial for catalysis.

The reduction of p-benzoquinone is catalyzed fairly efficiently by all species variants of ADH2, and this activity is not affected by the Pro47His mutation. However, it is expected that only small quinones and quinoneimines will be substrates since the substrate pocket only allows larger substituents in the para-position.

It is concluded that the ADH fold is suitable for generation of functional diversity, both in terms of isoenzyme divergence and mechanistic multiplicity.