Alcohol dehydrogenase (ADH) refers to a family of enzymes which catalyse the reversible oxidation of primary or secondary alcohols to aldehydes or ketones respectively. ADH has many roles in the body, a major function is to catalyse the oxidation of ethanol to acetaldehyde as the first step of ethanol metabolism by the liver, using NAD+ or NADP+ as electron acceptors in the process. Figure 1 shows ADH bound to ethanol with NAD binding sites occupied. However, ethanol is not the only target of these enzymes. Retinol, steroids and fatty acid molecules are also affected; different forms of ADH can oxidise primary, secondary, cyclic secondary, and even hemi-acetal alcohols. ADH catalysis occurs in the cell cytosol and in humans, converting ethanol to acetaldehyde, it has a turnover number of 1200 molecules per second. Researchers have currently discovered more than nine different varieties and humans often have six or more different types of alcohol dehydrogenase enzyme. Though these subtypes differ in aspects such as amino acid sequence, they catalyse the same reaction, though they vary in efficiency. ADH enzymes are found in almost every living organism as ethanol is a prominent organic compound present naturally in the environment due to processes such as fermentation, for example there is usually a small amount of alcohol in ripe fruit. Although the reaction is reversible, some types of alcohol dehydrogenase favour the forward reaction, while others favour the reverse.
The first ADH to be isolated was from brewer’s yeast or Saccharomyces cerevisaie in 1937 by Negelein and Wuluff. In 1948 Bonnichsen and Wassen then succeeded in crystallising ADH from horse liver. The properties of the animal and bacterial ADH were found to vary massively; the yeast enzyme is twice the size of animal ADH and is 100 times more active. The yeast enzyme is also far more specific. ADH was also one of the first enzymes to have its amino acid sequence and 3D structure determined, making it an important feature in the development of protein sequencing technologies.
There are more than nine different forms of ADH produced by the human body and each has a specific role in different areas of the digestive tract though the enzyme has been found to be especially highly concentrated in the liver and kidney. Though these forms have slightly varying structures, they also have many identical characteristics:
All forms of ADH have a common zinc domain per sub-unit formed from specific amino acid residueswhich serves to electrostatically stabilise the oxygen molecule of the alcohol group of ethanol. The zinc ion is co-ordinated to the sulphur atoms of two cysteine residues and the nitrogen atom of a histidine residue, as is shown by Figure 2.
The NAD+ binding domain is also a crucial feature of the enzyme as this is a cofactor required for reaction and is linked to the ethanol binding site by an alpha helix.
The zinc atom binds to the substrate via the oxygen of the carbonyl group. The purpose of the zinc ion is to polarise the carbonyl group of a substrate, hence favouring the transfer of a hydride ion from NADH. The way in which the zinc ion electrostatically stabilises the oxygen of ethanol serves to increase the acidity of the proton of the alcohol group. The histidine residue shown in figure 2 is then active by general base catalysis, allowing it to accept a proton from NADH, which in turn draws a proton from a nearby threonine residue. This results in a negatively charged threonine following the proton transfers, allowing the residue to accept a proton from the alcohol groupof the substrate. Simultaneously there is a hydride transfer to the NAD in its specific hydride accepting region. The result is the transfer of a hydride ion to NAD and the oxidation of the alcohol substrate to an aldehyde.
In bacteria ADH is vital for fermentation; an anaerobic cellular process in which organic food is converted into simpler compounds accompanied by the production of ATP. The reaction shown below is catalysed by ADH.
Glucose + 2Pi + 2ADP + 2H+ --> 2 ethanol + 2CO2 + 2 ATP + 2H20
Pyruvate formed from gylcolysis undergoes decarboxylation by pyruvate decarboxylase, again using NAD as a cofactor. NADH generated by the oxidation of glyceraldehyde-3-phosphate (G-3-P) is then converted to NAD+ during the reduction of acetaldehyde to ethanol. NADH is produced by the G-3-P dehydrogenase reaction. It is important for organisms which undergo fermentation to maintain a redox balance as both ADH and lactate dehydrogenase oxidise NADH to NAD+ so that the glycolytic pathway may continue.
In animals ethanol is primarily metabolised by the liver. The following reaction is the first step in ethanol metabolism and is catalysed by ADH.
CH3CH2OH + NAD+ --> CH3CHO + NADH + H+
The second step in ethanol metabolism involves aldehyde dehydrogenase and both of these steps produce NADH. If ethanol is consumed in excess then resulting high NADH concentrations act to inhibit gluconeogenesis. This, in turn, causes the reverse conversion of pyruvate to lactate which results in a build up of lactate. Excess lactate can be especially dangerous as it can potentially result in hypoglycaemia or lactic acidosis which effectively changes the pH of the blood. An excess of NADH will also inhibit fatty acid oxidation and, in fact, promotes the synthesis of fatty acids which can result in conditions such as alcoholic steatosis, or fatty liver.
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