Effects of substrate structure and temperature on the stereospecificity of secondary alcohol dehydrogenase from Thermoanaerobacter ethanolicus

Article abstract of DOI:10.1021/ja00165a057

Enzymes from thermophilic microorganisms are of considerable interest in biotechnological applications. The temperature dependence of the rates of reaction of the enantiomers of secondary alcohols with a secondary alcohol dehydrogenase (SADH) from a thermophilic bacterium, Thermoanaerobacter ethanolicus, has been determined. These results demonstrate a novel temperature-dependent reversal of stereospecificity. At temperatures below 26 °C, (S)-2-butanol is a better substrate than (R)-2-butanol on the basis of k(cat)/K(m) values; however, above 26 °C, (R)-2-butanol is a better substrate than (S)-2-butanol. (S)-2-Pentanol is the preferred substrate up to 60 °C; however, our data predict that (R)-2-pentanol would be preferred above 70 °C. (S)-2-Hexanol is predicted to be the preferred enantiomer up to 240 °C. At 50 °C, the k(cat)/K(m) values for oxidation of S alcohols obey a linear free energy relationship with respect to increasing chain length, indicating a constant mechanism. However, the R alcohols show a convex plot, suggesting that there is a chain-length-dependent change in the rate-determining step. 3-Pentanol, cyclobutanol, and cyclopentanol are also good substrates on the basis of comparison of their k(cat)/K(m) values with those of 2-propanol.