Yeast alcohol dehydrogenase and

It is worth noting that finding a secondary a-deuterium KIE larger than the EIE is not unique. In fact, it has been found in several other reactions. For instance, Cleland and co-workers (Cook et al., 1980,1981 Cook and Cleland, 1981a,b) found unexpectedly large secondary a-deuterium KIEs in some enzymatic reactions for example, a secondary a-deuterium KIE of 1.22 for the reduction of acetone catalysed by yeast alcohol dehydrogenase and a KIE of 1.34 for the reduction of cyclohexanone catalysed by horse-liver dehydrogenase. 

In the following year, Cleland and his coworkers reported further and more emphatic examples of the phenomenon of exaltation of the a-secondary isotope effects in enzymic hydride-transfer reactions. The cases shown in Table 1 for their studies of yeast alcohol dehydrogenase and horse-liver alcohol dehydrogenase would have been expected on traditional grounds to show kinetic isotope effects between 1.00 and 1.13 but in fact values of 1.38 and 1.50 were found. Even more impressively, the oxidation of formate by NAD was expected to exhibit an isotope effect between 1.00 and 1/1.13 = 0.89 - an inverse isotope effect because NAD" was being converted to NADH. The observed value was 1.22, normal rather than inverse. Again the model of coupled motion, with a citation to Kurz and Frieden, was invoked to interpret the findings. 

When the configuration of the ethanol- 1-d is inverted by conversion to the tosylate followed by treatment with hydroxide and the inverted ethanol-l-d is then oxidized with yeast alcohol dehydrogenase and NAD+, the deuterium (which has taken the stereochemical position of the original hydrogen) is now removed and the product is unlabeled CH3CH=0.28 The sequence of events121 s is summarized in Fig. 55. 

Conventional laboratory oxidation of the monodeuteriated ethanol 24 gives ethanal (acetaldehyde), which contains 50% of the deuterium present in 24 (Scheme 8.1). However, enzymes are chiral, and enzymic oxidation (by yeast alcohol dehydrogenase and NAD+) removes exclusively in the oxidation of ethanol to ethanal HR is labelled as D in Scheme 8.2. Clearly, the molecule of ethanol is presented to the chiral enzyme so as to form a unique diastereoisomeric complex in which only the proton can be removed in the oxidative elimination. 

Nicotinamide-(S-methylmercury-thioinosine) dinucleotide was found to exhibit coenzyme properties with lactate dehydrogenase and liver alcohol dehydrogenase, but inactivate yeast alcohol dehydrogenase and glyceraldehyde 3-phosphate dehydrogenase an essential thiol group was therefore modified in the last two cases. 

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. 

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