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15 14:04:11 +08'00'
A Toast to Alcohol Dehydrogenase
Jason R. Roque De La Salle University – Dasmariñas Dasmariñas City, Cavite, Philippines
INTRODUCTION Alcohol Dehydrogenase is a group of dehydrogenase enzymes that occur in many organisms. This type of dehydrogenase enzyme facilitates the interconversion between alcohols and aldehydes or ketones with the reduction of nicotinamide adenine dinucleotide (NAD+ to NADH). The breaking down of alcohols which is very toxic serves a major role in humans and other animals. They also participate in generation of useful aldehyde, ketone, or alcohol groups during biosynthesis of various metabolites. In yeast, plants and many bacteria, some alcohol dehydrogenases catalyze the opposite reaction as part of fermentation to ensure a constant supply of NAD+. (1) This is an enzyme that makes it possible for humans to drink beer, wine, and other alcoholic beverages. However, the true function of this enzyme is thought to be the conversion of alcohol generated by bacteria in the intestine to other metabolic products. Individuals with some mutant forms of ADH may be especially sensitve to alcohol. The molecule is a dimer made either of two identical or two different chains. (2) “Alcohol dehydrogenase is our primary defense against alcohol, a toxic molecule that compromises the function of our nervous system. The high levels of alcohol dehydrogenase in our liver and stomach detoxify about one stiff drink each hour. The alcohol is converted to acetaldehyde, an even more toxic molecule, which is then quickly converted into acetate and other molecules that are easily utilized by our cells. Thus, a potentially dangerous molecule is converted, through alcohol dehydrogenase, into a mere foodstuff.” (Goodsell, David. Molecule of the Month, 3)
STRUCTURE There are at least nine different forms of alcohol dehydrogenase that our bodies create. Each of them has their own distinct characteristics and properties. Almost all the mentioned forms of alcohol dehydrogenase are found primarily in the liver. The beta3 form and the other similar enzyme are found in the horse liver.
Figure 1. Human beta3 Alcohol Dehydrogenase (Davis, G.J et al., 1996)
Figure 2. Triclinic Ternary Complex of Horse Liver Alcohol Dehydrogenase (Eklund, H. et al., 1981) “The sigma form is found in the lining of the stomach. Each enzyme is composed of two subunits, and quite remarkably, you can mix and match subunits between these different forms, creating mixed dimers that are still active. Ethanol is not the only target of these enzymes, they also make important modifications to retinol, steroids, and fatty acids. The range of different types of alcohol dehydrogenase ensures that there will always be one that is perfect for the current task.” (Goodsell, David. Molecule of the Month, 3)
Figure 3. Human Sigma Alcohol Dehydrogenase (Xie, P. et al., 1997) Alcohol dehydrogenase is a homodimer. Each monomer has 374 residues with molecular weight of 74000 dalton. There are two domains. The NAD+-binding domain (residues 176-318) consists of a central beta-sheet of 6 strands flanked by alpha helices. NAD+ binds to the C-terminus of the beta-sheet. The catalytic domain (residues 1-175, 319-374) also has a alpha/beta structure. The inter-domain interface forms a cleft which contains the active catalytic site. The interface is formed by two helices, one from each domain crossing over each other. There are two Zn++ cations per monomer, one at the catalytic site being mandatory for catalysis. The alcohol substrate binds inside the cleft where the Zn++ cation is, whilst the nicotinamide ring of the NAD finds its way pointing into the cleft. The dimer forms with the two NAD-binding domains packing together such that their 2 central beta sheets combine to form a 12-stranded beta sheet.
Figure 4. Structure of ADH. (Goodsell, 2010)
MECHANISM OF CATALYST In the mechanism of alcohol dehydrogenase two molecular tools are used to perform the reaction with ethanol. Zinc atom, which is the first molecule, is used to have the alcoholic group of ethanol positioned and placed in the right order. The second molecule is a large NAD cofactor. This co-factor is constructed by the vitamin niacin. The NAD co-factor performs the reaction. The ADC molecule, which contains ethanol molecules, is bound to the two active sites. “A slightly-modified version of NAD was used in the structure analysis, so that the enzyme would not immediately attack the ethanol.” (Goodsell, David. Molecule of the Month, 3) You can see that the zinc atom, illustrated in the light blue, this is Notice how the zinc atom, shown in light blue, is supported by three amino acids from the protein: cysteine 46 to the left, cysteine 174 to the right, and histidine 67 above. The ethanol, illustrated as green and magenta, connects to the zinc and is placed next to the NAD cofactor, which broadens below the ethanol molecule in this (3) illustration.
Figure 5. Alcohol Dehydrogenase Reaction (Goodsell, 2010)
The class which Alcohol Dehydrogenase belong utilizes the same basic mechanism to form aldehydes or ketones from alcohol.
Figure 6. Mechanism of Alcohol to form Aldehydes or Ketones (4) ADH catalyzes the oxidation of alcohols by reducing NAD with a hydride. ADH also utilizes a zinc ion to electrostatically stabilize the alcohol oxygen, thus increasing the acidity of the alcohol’s proton. In the pathway, His 51 is activated by general base catalysis such that the histidine can then accept a proton from the NAD, which is turn draws a proton from Thr 48, again demonstrating general base catalysis (although this is rather indirect since the substrate has not yet been involved). These proton transfers ready the threonine (which is negatively
charged due to proton transfer to the NAD) for accepting a proton from the alcohol of the actual substrate (cyclohexanol in the case of this particular model). This is the first example of true base catalysis actually involving the substrate. At the same time, since this oxidation is concerted, there is a hydride transfer to the NAD in its traditional hydride accepting region. Thus, the whole sequence essentially amounts to transfer of hydride to the NAD and the oxidation of an alcohol to an aldehyde. Key points are the orientation of the amino acid proton acceptors and donors, as well as the position of the zinc ion in relation to the substrate such that it stabilizes a negative charge on the substrate thereby taking part in transition state stabilization. (4) The Mechanism for alcohol dehydrogenase follows a random bisubstrate mechanism. In the mechanism, the NAD+ and alcohol bind to the enzyme, so that the enzyme is now attached to the two substrates. While attached, the hydrogen is formally transferred from the alcohol to NAD, resulting in the products NADH and a ketone or aldehyde. The two products are then released, and the enzyme has catalyzed the reaction. (5)
KINETICS OF REACTION The alcohol dehydrogenase catalyzed aldehyde-NADH reaction show kinetics consistent with a random-order mechanism, and the rate-limiting step is the dissociation of the product enzyme-NAD+ complex. Alcohol dehydrogenase is more effective for smaller alcohol substrates, and it becomes less effective as substrate size increases. It is also more effective for primary than secondary alcohols.(6) In a study where ADH was immobilized in tresyl-chlorideactivate agarose, it was shown that the Michaelis-Menten model could not take into consideration all the constraints induced by the immobilization on the enzyme properties but that the Theorell-Chance model was more appropriate.(7) The rate of an enzyme catalysed reaction is determined by the number of enzyme molecules which are binding to the substrate; i.e. the concentration of the enzyme-substrate complex. NAD+ + ADH ethanol + NAD+ADH complex ethanol: AD+:ADH complex ethanal: NADH: ADH complex NADH:ADH complex NAD+ADH complex ethanol: AD+:ADH complex ethanal: NADH: ADH complex ethanal + NADH:ADH complex NADH + ADH
Inhibitors reduce the reaction velocity by combining with the enzyme but not reacting. 2,2,2-Trifluoroethanol (CF3CH2OH) has a similar structure to ethanol. Although it binds strongly, it cannot react due to the electron-withdrawing nature of the highly electronegative fluorine atoms, which hold the electrons in the C-H bonds and will not allow them to transfer (as a hydride ion) to the NAD+.
NAD+ + ADH
trifluoroethanol + NAD+ADH complex trifluoroethanol:NAD+:ADH complex trifluoroethanol:NAD+:ADH complex no reaction Both trifluoroethanol and ethanol compete for the enzyme, dependent on their concentration and inherent attraction (binding affinity). The process is reversible and the inhibitor may be removed from the enzyme by excess substrate. Thus at a fixed trifluoroethanol (inhibitor) concentration, the percentage inhibition falls as the concentration of ethanol [substrate] rises.
APPLICATIONS AND CLINICAL SIGNIFICANCES In fuel cells, alcohol dehydrogenases can be used to catalyze the breakdown of fuel for an ethanol fuel cell. Scientists at Saint Louis University have used carbon-supported alcohol dehydrogenase with poly(methylene green) as an anode, with a nafion membrane, to achieve about 50 μA/cm2.(8) In biotransformation, alcohol dehydrogenases are often used for the synthesis of enantiomerically pure stereoisomers of chiral alcohols. In contrast to the chemical process, the enzymes yield directly the desired enatiomer of the alcohol by reduction of the corresponding ketone. Alcoholism There have been studies showing that ADH may have an influence on the dependence on ethanol metabolism in alcoholics. Researchers have tentatively detected a few genes to be associated with alcoholism. If the variants of these genes encode slower metabolizing forms of ADH2 and ADH3, there is increased risk of alcoholism. The studies have found that mutations of ADH2 and ADH3 are related to alcoholism in Asian populations. However, research continues in order to identify the genes and their influence on alcoholism. (9) Drug dependence Drug dependence is another problem associated with ADH, which researchers think might be linked to alcoholism. One particular study suggests that drug dependence has seven ADH genes associated with it. These results may lead to treatments that target these specific genes. However, more research is necessary. (10)
(1) Miller, F. P., Vandome, A. F., & McBrewster, J. 2010. Alcohol Dehydrogenase. VDM Publishing House Ltd. (2) Alcohol Dehydrogenase. Retrieved 29 August 2011 from http://whozoo.org/mac/Music/ADH.htm
(3) Goodsell, David. Molecule of the Month (January 2001): Alcohol Dehydrogenase. Retrieved 30 August 2011 from http://www.pdb.org/pdb/101/motm.do?momID=13 (4) Alcohol Dehydrogenase. Retrieved 30 August 2011 from http://www.users.csbsju.edu/~hjakubow/classes/rasmolchime/99ch331proj/alcohold ehydro/index.htm (5) Voet, et. al. Fundamentals of Biochemistry: 3rd Edition. Hoboken: Wiley & Sons, Inc, 2008. (6) Dickinson FM, Monger GP. A study of the kinetics and mechanism of yeast alcohol dehydrogenase with a variety of substrates. Retrieved 30 August 2011 from http://www.ncbi.nlm.nih.gov/pubmed/4352908 (7) Bille V, Remacle J. Simple-kinetic descriptions of alcohol dehydrogenase after immobilization on tresyl-chloride-activated agarose. Retrieved 30 August 2011 from http://www.ncbi.nlm.nih.gov/pubmed/3769934 (8) Moore CM, Minteer SD, Martin RS (February 2005). "Microchip-based ethanol/oxygen biofuel cell" (9) Sher KJ, Grekin ER, Williams NA (2005). "The development of alcohol use disorders”. Annual Review of Clinical Psychology (10) Luo X, Kranzler HR, Zuo L, Wang S, Schork NJ, Gelernter J (February 2007). "Multiple ADH genes modulate risk for drug dependence in both African- and EuropeanAmericans".Human Molecular Genetics
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