EFFECTS OF SUBSTRATE STRUCTURE AND TEMPERATURE ON THE STEREOSPECIFICITY OF SECONDARY ALCOHOL-DEHYDROGENASE FROM THERMOANAEROBACTER-ETHANOLICUS

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 kcat/Kmvalues; 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 kcat/Kmvalues 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 kcat/Kmvalues with those of 2-propanol. © 1990, American Chemical Society. All rights reserved.