Alcohol dehydrogenase is a part of the oxidoreductase family which catalyzes the oxidation of alcohols using NAD+Â or NADP+Â as the electron acceptor. It has a reversible reaction and its substrates can be a variety of primary or secondary alcohols, and hemiacetals (1). A toxic molecule that compromises the function of our nervous system is our primary defense against alcohol. The high levels of alcohol dehydrogenase in our liver and stomach detoxify about one stiff drink each hour. An even more toxic molecule is then converted into acetate and other molecules that are easily utilized by our cells using alcohol that is converted into acetaldehyde. (2).
In 1937, the yeast by Negelein and Wulff was first purified and crystallized from brewers from alcohol dehydrogenase. Bonnichsen and Wassen crystallized ADH from horse liver in 1948. It was found that these two ADHs differed in many of their properties. In the early 1950s Theorell and Chance studied the stoichiometry and dissociation constant of the mammalian enzyme complex.Â It was found that the yeast enzyme is twice the size of mammalian ADH, and approximately 100 times more active. In 1955, Vallee and Hoch confirmed the presence of Zinc metal. In the 1960s the inhibitors of alcohol dehydrogenase were studied along with the roles of specific structural components. Structural and kinetic studies continued on into the 1970s when conformational changes associated with binding were investigated, along with the isozymes. During the 1980s the genetics, biochemistry, and developmental regulation of ADH in various species, including mice and pig, were investigated. The 1990s brought better understanding of the role of the zinc metal, and the discovery of additional inhibitors. Recent research has focused on obtaining alcohol dehydrogenases with higher catalytic activity, and a better understanding ADH gene regulation (1).
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Alcohol dehydrogenase (ADH) is a part of the oxidoreductase family which catalyzes the oxidation of alcohols using NAD+Â or NADP+Â as the electron acceptor. It has a reversible reaction and its substrates can be a variety of primary or secondary alcohols, and hemiacetals (1).
Figure 1. Structure of Alcohol Dehydrogenase
Horse liver alcohol dehydrogenase is commonly studied due to its structural homology to the human enzyme and the availability of sample. The following information relates to the horse LADH unless otherwise noted. The active form of LADH is a homodimer with a combined molecular weight of 80 kDa with two subunits 374 amino acid residues long, each containing its own catalytic binding domain. The monomer secondary structures are made up of 9 Î±-helices, including two around the active site, an Î±/Î² parallel twisted sheet motif composed of six sheets, and an antiparallel Î²-sheet motif in a twisted type arrangement on the exterior of the structure. The two monomers are joined together by an overlapping region in the Î±/Î² parallel sheet in which a loop and helix from one monomer extends across to the other monomer. There is also a partial overlap between the respective parallel Î²-sheets in which the alignment of the sheets is antiparallel between monomers in the overlap. There are two zinc atoms in each subunit with one involved in the reaction. The catalytic zinc is tetra coordinated in the active site by residues Cys-46, Cys-174, His-67 and by the substrate hydroxyl or water molecule when substrate is not bound. The other zinc atom is located in a loop region near the exterior, is coordinated by four cysteine residues (Cys-97, 100, 103, and 111), and plays no role in catalysis but rather appears to have a role in structural stability. LADH also contains the cofactor NAD+ which acts as the redox partner in the reaction by being reduced to NADH. The NAD+ molecule sits in a binding cleft with the carboxyl end loops of the Î±/Î² parallel sheet on one side of it and the active site on the opposite side. For the substrate to bind to the active site it must first pass through a hydrophobic tunnel-like region in which the active site zinc atom sits at the end of the tunnel (3).
Figure 2. Structure of LADH dimer
Mechanism of Catalysis, Kinetics of Reaction and Mode of Regulation
The reaction mechanism for alcohol dehydrogenase has been studied extensively since it was first hypothesized in the 1950's and the data has shown that the reaction follows a specific order of binding in which NAD+ is first to bind, followed by the substrate alcohol, then release of the oxidized ketone or aldehyde species, and finally the dissociation of the reduced NADH coenzyme. The redox reaction that takes place occurs with two distinct steps that will be discussed in greater detail; (1) the deprotonation of the alcohol to generate zinc bound alkoxide ion, and (2) a hydride transfer reaction between the substrate Î±-carbon and the coenzyme to complete the double bond formation in the substrate and formation of the reduced NADH species. This mechanism shares some similarity with other dehydrogenase enzymes that utilize NAD+ as a redox partner in both the specific binding sequence and the hydride transfer and deprotonation steps of the reaction (3).
Figure 3. Mechanism of ADH catalysis
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The first reaction step is Deprotonation. Once the coenzyme and substrate have bound to the proper orientation in the active site, the first step in the redox reaction is the extraction of a proton from the hydroxyl of the substrate by the Ser-48 residue of LADH. This generates an alkoxide ion which forms a complex with the active site zinc to give stability to the highly reactive anion so that it does not extract a proton from a nearby residue and revert back to the alcohol species. Without this stabilization from the zinc it is estimated that the activity of the enzyme would be diminished by a factor of about 100 fold, indicating that the metal ligand is extremely important in the reaction mechanism through its stabilization of the intermediate. The extracted proton is then transferred via a hydrogen bond network from Ser-48, then to the C2 hydroxyl of the nicotinamide ribose ring, on to the C3 hydroxyl of the ribose, and then finally to the imidazole nitrogen of residue His-51. It is important to note that there is not an actual movement of a single hydrogen atom from the alcohol onto His-51 but rather the hydrogen bonding network between all of these species forms a type of extended intermediate structure. In essence it acts as a type of proton exchange system in which each species binds the hydrogen from the former species and releases its hydrogen to the latter species until the exchange has moved from the substrate to His-51. Though structural data has led to a general agreement on the proton transfer mechanism from the substrate to His-51, the mechanism of proton transfer from His-51 to the solvent is unclear. One potential pathway for this to occur is via a water molecule that is hydrogen bonded to both His-51 and Ser-54. A transfer through this molecule would lead the proton transfer to occur though several water molecules in the substrate binding pocket and ultimately out to the solvent]. Another proposed mechanism involves a rotation of His-51 by approximately 20Â° about the CÎ±-CÎ² bond to allow proton transfer with a water molecule that is hydrogen bonded to the carbonyls of residues 270 and 294. This mechanism leads to a transfer between networks of water molecules out a channel (not the substrate binding pocket) to the solvent. Though the exact pathway of the final step is unknown, the net effect of this process is a removal of a proton from the substrate which is transferred out to the solvent as the first half of the oxidation of the alcohol to a ketone/aldehyde (3).
Figure 4. Mechanism of Proton Transfer Reaction
The second step in the oxidation is a direct hydride transfer between the newly formed alkoxide ion and the NAD+ coenzyme. This hydride transfer is characterized by homolytic cleavage of the alkoxide carbon hydrogen bond in which a proton and two electrons are transferred across a short distance from substrate to coenzyme. The pro-R donor hydrogen from the substrate has been shown by pentafluorobenzyl alcohol structural data to be located about 3.4 Å away from the acceptor C4 of the nicotinamide ring and is pointing directly at it. The hydride transfer is accompanied by the formation of a double bond in the alkoxide ion to complete the oxidation to the appropriate ketone/aldehyde species. Upon forming the oxidized species, the LADH enzyme binding affinity for the substrate is greatly reduced and preferentially binds a water molecule. Coordination of water to the zinc atom kicks out the product ketone/aldehyde through the substrate binding pocket and into the solvent. After product release there is a dissociation of the reduced NADH cofactor which concludes the catalytic cycle. An interesting aspect of the hydride transfer is that it has been shown to exhibit some hydrogen tunneling effects in the oxidation of benzyl alcohol in the horse liver and yeast alcohol dehydrogenase enzymes (3).
Alcohol dehydrogenase is a useful enzyme found in humans and most organisms to catalyze redox reactions with various alcohol substrates. One of the most fascinating aspects about LADH is that it utilizes simple chemical interactions facilitated by intricate structural positioning to perform an oxidation/reduction reaction without the use of an energy molecule to drive the process. Cofactor and substrate bind to form a complex driven by hydrogen bonding interactions and ion pairing within their binding sites. A dimeric protein structure composed of over 700 residues undergoes a conformational change because of an imbalance caused by the addition of the new ligands. The substrate hydroxyl coordinates with a bound zinc atom and loses a proton to a near perfectly aligned and spaced relay system of residues which completes a transfer all the way to the solvent. A hydride transfer reaction driven by a quantum mechanical tunneling phenomenon forms the oxidized product and reduced coenzyme species, which bind less efficiently than their substrate counterparts and thus dissociate from the complex. The process completed, the enzyme becomes available to repeat the cycle again and again. This vast composite of simple chemistry demonstrates the simultaneous power and elegance of enzymes. Finely tuned by millions of years of biological selection, these proteins are vital components of living organisms because of their unique ability to use three dimensional structural forces to bring reactive species into a spacing and geometry that facilitates a reaction much more efficiently than in solution (3).
Associated Diseases and Importance of ADH to Human Health
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To counter a common toxin in our environment, Alcohol dehydrogenase gives a line of defense. But not all protection is good, some also carries danger. ADH modifies other alcohols which produces dangerous products. Example is methanol, which is commonly used to "denature" ethanol. Formaldehyde is used to convert alcohol dehydrogenase. The formaldehyde does the damage, attacking proteins and embalming them. Small amounts of methanol cause blindness, as the sensitive proteins in the retina are attacked, and larger amounts, lead to damage and death (2). A particular alcohol dehydrogenase, polymorphism (allele A1) in the promoter region of the gene has been recently demonstrated to be associated with increased risk of Parkinson's disease (PD) (4). However, this disease involving ADH is still subjected to further studies.