Alcohol dehydrogenase catalytic mechanism

Fig. 3. Carbocation- and alkoxide ion-like structures of the reactive species postulated to be involved in the hydride transfer step of the horse liver alcohol dehydrogenase catalytic mechanism.

The problem of biomimetic model design simulating the action mechanism of corresponding enzymes is based on the idea of structural-functional conformity. In 1971, alcohol dehydrogenase was primarily synthesized [123], In this biomimetic system the product is formed due to direct electron transfer from the reduced co-factor (NADH) analog to aldehyde. Note that the display of alcohol dehydrogenase catalytic activity requires the presence of zinc (II) ion. 

Uncovering of the three dimentional structure of catalytic groups at the active site of an enzyme allows to theorize the catalytic mechanism, and the theory accelerates the designing of model systems. Examples of such enzymes are zinc ion containing carboxypeptidase A 1-5) and carbonic anhydrase6-11. There are many other zinc enzymes with a variety of catalytic functions. For example, alcohol dehydrogenase is also a zinc enzyme and the subject of intensive model studies. However, the topics of this review will be confined to the model studies of the former hydrolytic metallo-enzymes. 

NADH. The enzymes are widely distributed in nature, being found in microorganisms, plants, and animals. Catalytic mechanism, specificity, and physical properties of the alcohol dehydrogenases have been reviewed in detail (91-93). 

While most alkaloids do not contain aldehydes when they enter mammalian, microbial, or plant tissues, this functional group may become important when formed as a metabolite of alcohols (via alcohol dehydrogenase) or amines (via oxidative dealkylation and oxidative deamination). Aldehyde dehydrogenases catalyze oxidation of aldehydes to the corresponding carboxylic acids. The physical properties, catalytic mechanism, and specificity of this group of enzymes has been reviewed (99). The general reaction catalyzed by aldehyde dehydrogenase is seen in Eq. (9). 

This zinc metalloenzyme [EC 1.1.1.1 and EC 1.1.1.2] catalyzes the reversible oxidation of a broad spectrum of alcohol substrates and reduction of aldehyde substrates, usually with NAD+ as a coenzyme. The yeast and horse liver enzymes are probably the most extensively characterized oxidoreductases with respect to the reaction mechanism. Only one of two zinc ions is catalytically important, and the general mechanistic properties of the yeast and liver enzymes are similar, but not identical. Alcohol dehydrogenase can be regarded as a model enzyme system for the exploration of hydrogen kinetic isotope effects. 

The catalytic activity of liver alcohol dehydrogenase is strongly pH dependent over a wide range. It has been well established that this pH dependence derives from the combined effects of pH on several steps in the catalytic mechanism. They are all proton equilibria involving... 

Catalytic Mechanism of Horse Liver Alcohol Dehydrogenase, a Medium-Chain Dehydrogenase... 

Youn, B., Camacho, R., Moinuddin, S.G.A., Lee, C., Davin, L.B., Lewis, N.G., Kang, C.H. (2006a) Crystal structures and catalytic mechanism of the Arabidopsis cinnamyl alcohol dehydrogenases AtCADS and AtCAD4. Org. Biomol. Chem., 4,1687-97. 

NO-induced protein S-glutathionylation was proposed for the first time in 1988 by J.W. Park as a possible mechanism for the inactivation of yeast alcohol dehydrogenase by NO [32]. However, it took almost 10 years until the possibility that NO might be able to direct the incorporation of GSH to protein sulfhydryls was reconsidered. In 1997. it could be demonstrated that micromolar concentrations of GSNO inhibit aldose reductase through site-specific mixed disulphide formation at a conserved cysteine residue in its catalytic site... 

O. Tapia, H. Eklund, and C.I. Branden, "Molecular, Electronic, and Structural Aspects of the Catalytic Mechanism of Alcohol Dehydrogenase, in Steric Aspects of Biomolecular Interactions, G. Ndray-Szabd and K. Simon (Eds.), CRC Press, West Palm Beach, 1987. 

Alcohol dehydrogenase contains two atoms of zinc. One atom participates in the catalytic mechanism, while the other serv es a structural role. The enzyme actually is a dimer, and is composed of two subuiuts. Several types of alcohol dehydrogenase occur in the body. The enzymes have closely related amino acid sequences, and thus consist of a family of related enzymes. 

Analytical and enz5onological data lead to the conclusion that zinc is a structural and functional component of ADH, and that it participates in the mechanism of its enzymatic action (Vallee and Hoch, 1955b). The four metal atoms are firmly bound to the protein apoenzyme. Pending the results of investigation in progress, this discussion will assume that the four zinc atoms are bound to the protein in an equivalent manner, and that each atom of zinc acts independently of the other three in the catalytic action of alcohol dehydrogenase. This seems the more acceptable view at present, since Hayes and Velick (1954) found the four binding sites of DPN to be equivalent. 

Zhang Y, Huang X, Li Y (2008) Negative effect of [bmim][PFJ on the catalytic activity of alcohol dehydrogenase mechanism and prevention. J Chem Technol Biotechnol 83 1230-1235... 

The tris(2-mercapto-l-mesitylimidazolyl)borate ligand, TmMs, reacts with Zn(C104)2-6H20 yielding [Zn(TmMs)(HOMe)]+, a stable monomeric tetrahedral zinc-methanol complex which shows analogies with the alcohol intermediate in the catalytic cycle of the mechanism of action of liver alcohol dehydrogenase.57... 

The fact that enzymes employ dynamics, should in no way be surprising -evolution knows nothing of quantum mechanics, classical mechanics, or vibration-ally enhanced tunneling. Rates of reaction are optimized for living systems using all physical and chemical mechanisms available. It is also important to point out that such protein dynamics are far from the only contributor to the catalytic effect. In fact in an enzyme such as alcohol dehydrogenase, transfer of a proton from the alcohol to the coordinated zinc atom is critical to the possibility of the reaction. The specific modulation of the chemical barrier to reaction via backbone protein dynamics is now seen to be part of the chemical armamentarium employed by enzymes to catalyze reactions. 

CAD is a type A reductase, abstracting the prol hydride from NADPH via a two-electron hydride transfer mechanism. It belongs to the alcohol dehydrogenase (ADH) family, the members of which are zinc-dependent medium-chain dehydrogenases/reductases (MDR) with two Zn ions per subunit. One zinc atom is thought to have a structural role, whereas the other forms the core of the catalytic site. Interestingly, medium-chain zinc-dependent ADHs exist as homotetramers as in bacteria, archaea, and yeast, or as homodimers as in plants and vertebrates. 

In an alternative mechanism, the substrate molecule is again coordinated to tetrahedral Zn, with the coordinated water molecule now serving as a site for transient proton transfer, thereby generating a penta-coordin ated zinc intermediate. Formation of this intermediate during substrate turnover was supported by time-resolved freeze-quenched X-ray absorption fine spectroscopic analysis of the thermophilic bacterium Thermoanaerobaaer brockii alcohol dehydrogenase (TbADH). These results thus provided further evidence for the dynamic alteration of the Zn from a tetrahedral to a penta-coordinated form with detection of two new penta-coordinated intermediate states these included the water molecule in the zinc coordination sphere during a single catalytic cycle. 

The enzyme also catalyzes the oxidation of aldehydes to acids irreversibly (65). With catalytic amounts of NAD complete dismutation to equivalent amounts of acid and alcohol occurs. The mechanism has been studied in some detail (66). The < 2 (and K ) values for aldehydes in the aldehyde dehydrogenase reaction, measured by proton production, are large, comparable to those for secondary alcohols, as would be expected if the hydrated forms of the aldehydes are the true substrates... 

Metalloenzymes are enzymes that have a tightly bound metal ion. These metal ions are normally incorporated into the enzymes during enzyme synthesis, and removal of the metal ions often results in the complete denat-uration of the enzyme. These metal ions may contribute either to the structure or the catalytic mechanism of a metalloenzyme. Lor example, horse liver alcohol dehydrogenase contains two tightly bound zinc ions (Zn ). The first zinc ion is structural it is bound to four cysteine side chains and is essential to maintain the structural integrity of the enzyme. The second zinc ion is catalytic it is bonnd to the side chains belonging to two cysteines and one histidine at the active site of the enzyme, and it participates in the catalytic cycle of the enzyme. 

---