Alcohol dehydrogenase enzyme activity

The chiral compounds (/ )- and (5)-bis(trifluoromethyl)phenylethanol are particularly useful synthetic intermediates for the pharmaceutical industry, as the alcohol functionality can be easily transformed without a loss of stereospecificity and biological activity, and the trifluoromethyl functionalities slow the degradation of the compound by human metabolism. A very efficient process was recently demonstrated for the production of the (5)-enantiomer at >99% ee through ketone reduction catalyzed by the commercially available isolated alcohol dehydrogenase enzyme from Rhodococcus erythropolis (Figure 9.1). The (7 )-enantiomer could be generated at >99% ee as well using the isolated ketone reductase enzyme KRED-101. 

The reduction of 3- and 4-thiepanones (41 and 42) was reported using either hydride (LAH) (67AG(E)872, 70JOC584) or horse liver alcohol dehydrogenase enzymes which gave the 3-hydroxy- (136) and 4-hydroxy- (43) thiepane in optically active form (81CJC1574. ... 

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. 

The Protein Data Bank PDB ID 1A71 Colby T D Bahnson B J Chin J K Klinman J P Goldstein B M Active Site Modifications m a Double Mutant of Liver Alcohol Dehydrogenase Structural Studies of Two Enzyme Ligand Com plexes To be published... 

Figure 1.9 Examples of functionally important intrinsic metal atoms in proteins, (a) The di-iron center of the enzyme ribonucleotide reductase. Two iron atoms form a redox center that produces a free radical in a nearby tyrosine side chain. The iron atoms are bridged by a glutamic acid residue and a negatively charged oxygen atom called a p-oxo bridge. The coordination of the iron atoms is completed by histidine, aspartic acid, and glutamic acid side chains as well as water molecules, (b) The catalytically active zinc atom in the enzyme alcohol dehydrogenase. The zinc atom is coordinated to the protein by one histidine and two cysteine side chains. During catalysis zinc binds an alcohol molecule in a suitable position for hydride transfer to the coenzyme moiety, a nicotinamide, [(a) Adapted from P. Nordlund et al., Nature 345 593-598, 1990.)...

In order to broaden the field of biocatalysis in ionic liquids, other enzyme classes have also been screened. Of special interest are oxidoreductases for the enan-tioselective reduction of prochiral ketones [40]. Formate dehydrogenase from Candida boidinii was found to be stable and active in mixtures of [MMIM][MeS04] with buffer (Entry 12) [41]. So far, however, we have not been able to find an alcohol dehydrogenase that is active in the presence of ionic liquids in order to make use of another advantage of ionic liquids that they increase the solubility of hydrophobic compounds in aqueous systems. On addition of 40 % v/v of [MMIM][MeS04] to water, for example, the solubility of acetophenone is increased from 20 mmol to 200 mmol L ... 

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. 

Hageman, R.H. Flesher, D. (1960). The effect of anaerobic environment on the activity of alcohol dehydrogenase and other enzymes of corn seedlings. Archives of Biochemistry and Biophysics, 87, 203-9. 

Figure 3. (A) Determination of molecular mass of pectic enzymes by gel filtration in Sepharose 6B. Molecular mass markers - tyroglobulin, 2- apoferritin, 3- p-amylase, 4-alcohol dehydrogenase, 5- bovine serum albumin, 6- carbonic anhydrase. (B) SDS-PAGE of pectolytic activities. Molecular mass markers 1- myosin, 2- p-galactosidase, 3- phosphorylase b, 4- bovine serum albumin, 5- ovalbumin, 6- carbonic anhydrase.

In addition to enzyme activity, the concentration of an nonelectroactive substrate can be determined electrochemically by this technique. By keeping the substrate (analyte) the limiting reagent, the amount of product produced is directly related to the initial concentration of substrate. Either kinetic or equilibrium measurements can be used. Typically an enzyme which produces NADH is used because NADH is readily detected electrochemically. Lactate has been detected using lactate dehydrogenase, and ethanol and methanol detected using alcohol dehydrogenase... 

Co-for-Zn substitution in alcohol dehydrogenase from Saccharomyces cerevisiae revealed a 100-fold increase in activity and a higher resistance of the modified protein to the inhibitory action of other divalent transition metals,1208 making the Co-modified enzyme suitable for biotechnological applications. 

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