Alcohol dehydrogenases from horse liver

G. B. Strambini, Singular oxygen effects on the room-temperature phosphorescence of alcohol dehydrogenase from horse liver, Biophys. J. 43, 127-130 (1983). 

N. Barboy and J. Feitelson, Quenching of tryptophan phosphorescence in alcohol dehydrogenase from horse liver and its temperature dependence, Photochem. Photobiol. 41, 9-13 (1985). 

Activation with alcohol dehydrogenase from yeast ° Activation with alcohol dehydrogenase from horse liver... 

The NAD+-dependent alcohol dehydrogenase from horse liver contains one catalytically essential zinc ion at each of its two active sites. An essential feature of the enzymic catalysis appears to involve direct coordination of the enzyme-bound zinc by the carbonyl and hydroxyl groups of the aldehyde and alcohol substrates. Polarization of the carbonyl group by the metal ion should assist nucleophilic attack by hydride ion. A number of studies have confirmed this view. Zinc(II) catalyzes the reduction of l,10-phenanthroline-2-carbaldehyde by lV-propyl-l,4-dihy-dronicotinamide in acetonitrile,526 and provides an interesting model reaction for alcohol dehydrogenase (Scheme 45). The model reaction proceeds by direct hydrogen transfer and is absolutely dependent on the presence of zinc(II). The zinc(II) ion also catalyzes the reduction of 2- and 4-pyridinecarbaldehyde by Et4N BH4-.526 The zinc complex of the 2-aldehyde is reduced at least 7 x 105 times faster than the free aldehyde, whereas the zinc complex of the 4-aldehyde is reduced only 102 times faster than the free aldehyde. A direct interaction of zinc(II) with the carbonyl function is clearly required for marked catalytic effects to be observed. 

Alcohol dehydrogenase-catalyzed reduction of ketones is a convenient method for the production of chiral alcohols. HLAD, the most thoroughly studied enzyme, has a broad substrate specificity and accommodates a variety of substrates (Table 11). It efficiently reduces all simple four- to nine-membered cyclic ketones and also symmetrical and racemic cis- and trans-decalindiones (167). Asymmetric reduction of aliphatic acyclic ketones (C-4-C-10) (103,104) can be efficiently achieved by alcohol dehydrogenase isolated from Thermoanaerobium brockii (TBADH) (168). The enzyme is remarkably stable at temperatures up to 85°C and exhibits high tolerance toward organic solvents. Alcohol dehydrogenases from horse liver and T. brockii... 

The availability of sufficient quantities of enzymes for crystallization studies has led to the crystal structures been obtained for several dehydrogenases. For example, two tetrameric NADP+-dependent bacterial secondary alcohol dehydrogenases from the mesophilic bacterium Clostridium beijerinckii and the thermophilic bacterium Thermoanaerobium brockii have been crystallized in the apo- and the holo-enzyme forms, and their structures are available in the Protein Data Bank11451. The crystal structure of the alcohol dehydrogenase from horse liver is also available[40 21. 

Oxidations Catalyzed by Alcohol Dehydrogenase from Horse Liver (HLADH)... 

It is noteworthy, here, that one conunercial source of purified alcohol dehydrogenase is horse liver. There is a considerable quantity of this enzyme in horse liver. It is likely that alcohol arises from fermentation in the caecum of the high content of fibre present in the food of the horse. Hence its liver will be exposed chronically to high concentrations of alcohol, and the liver adapts by synthesising large quantities of the enzyme. 

Alcohol dehydrogenase from pig liver (/ e-face selective), from Mucor javanicus (Si-face selective), and, especially, from horse liver (/fe-face selective) can be used for the reduction of cyclic ketones. The horse liver enzyme is very well characterized and models for the prediction of the stereochemical course of the reaction have been developed278,279. When subjected to horse liver alcohol dehydrogenase (HLADH) reduction, the following decalin derivatives yield products with >98% ee279,280. e.g. ... 

Using two types of specially synthesized rhodium-complexes (12a/12b), pyruvate is chemically hydrogenated to produce racemic lactate. Within the mixture, both a d- and L-specific lactate dehydrogenase (d-/l-LDH) are co-immobilized, which oxidize the lactate back to pyruvate while reducing NAD+ to NADH (Scheme 43.4). The reduced cofactor is then used by the producing enzyme (ADH from horse liver, HL-ADH), to reduce a ketone to an alcohol. Two examples have been examined. The first example is the reduction of cyclohexanone to cyclohexanol, which proceeded to 100% conversion after 8 days, resulting in total TONs (TTNs) of 1500 for the Rh-complexes 12 and 50 for NAD. The second example concerns the reduction of ( )-2-norbornanone to 72% endo-norbor-nanol (38% ee) and 28% exo-norbornanol (>99% ee), which was also completed in 8 days, and resulted in the same TTNs as for the first case. 

Fig. 35. Diagrammatic representation of functionally equivalent groups around the substrate in lactate dehydrogenase, glyceraldehyde-3-phosphate dehydrogenase and horse liver alcohol dehydrogenase. From the work of Rossmann and colleagues [164],...

Electrospray ionization has allowed the observation of a great number of non-covalent complexes protein-protein, protein-metal ion, protein-drug and protein-nucleic acid. About one-third of the proteins exist as multimeric forms. Mass spectrometry allows the study of their quaternary structure. This has been done for alcohol dehydrogenase (ADH) from horse liver and from yeast. The ESI spectra are displayed in Figure 8.22. The horse liver ADH is observed to be dimeric whereas that of yeast is tetrameric [131]. 

Horse liver alcohol dehydrogenase, HLADH, (also abbreviated as ADH or LADH) is the most extensively studied oxido-reductase. It plays a central role in ethanol metabolism and has been one of the main tools for understanding the mechanism of this process.15 it was crystaliized from horse liver in 1948 by Bonnichen and Wassen and is commercially available. Three isozymes EE, ES and SS are formed by dimeric combination of two different, E or S (E "ethanol-active" and S "steroid active"), protein chains. 16 The EE- isozyme of HLADH has been used in organic synthesis. 

Yeast alcohol dehydrogenase was crystallized from brewer s yeast by Negelein and Wulff (1937) and found to be dependent upon DPN for its activity by Anderson (1934). A distinctly different alcohol dehydrogenase was crystallized from horse liver by Bonnichsen and Wass6n (1948) and Bonnichsen (1950). The present discussion will be concerned primarily with those structural and functional aspects of the yeast enzyme which... 

Other enzymes, e.g., yeast alcohol dehydrogenase and alcohol dehydrogenase isolated from horse liver, are also used for this type of reduction26, however, the former is too specific for small molecules while the latter is more useful for the reduction of cyclic ketones. 

Fig. is. Range of substrates for the horse liver and Drosophila alcohol dehydrogenases (from left to right) secondary alcohols, primary alcohols, hemiacetals, and gem-diols. 

The importance of obstruction (steric hindrance) in determining the stereochemical course of enzymic reactions is further illustrated by a comparison of the stereospecificities of the alcohol dehydrogenases from yeast and from horse liver. The enzymes from both sources are A-side (re face) specific dehydrogenases wherein the pro-(R) hydrogen at C-l of the alcohol is in the transferring position Eq. (2)] ... 

Redox reactions catalyzed by alcohol dehydrogenases (e.g., from horse liver, HLADH) may be performed in organic solvents in both the reduction and oxidation mode, if the recycling system is appropriately modified (Sect. 2.2.1). Reduction of aldehydes/ketones and oxidation of alcohols is effected by NADH- or NAD" -recycling, using ethanol or wobutyraldehyde respectively. 

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