EFFECTS OF PH ON ENANTIOSPECIFICITY OF ALCOHOL DEHYDROGENASES FROM THERMOANAEROBACTER-ETHANOLICUS AND HORSE LIVER

Although there has been a great deal of interest in the effects of phy sical variables such as solvent and temperature on the stereochemistry of enzymatic reactions, there have been few studies of the effects of pH on the stereochemistry of enzymatic reactions. Furthermore, a theo retical framework for prediction and analysis of the effects of pH on the stereochemical course of enzymatic reactions has been lacking. We have now developed a theoretical model for the effects of pH on enzyma tic stereochemistry based on differential catalytic commitments for en antiomeric substrates. This model predicts that enantiomeric substrate s may show different apparent pK(a)s in their pH dependence of k(cat)/ K-m. In order to test this model, we have examined the effects of pH o n the reactions of both horse liver alcohol dehydrogenase (HLADH) and secondary alcohol dehydrogenase (SADH) from Thermoanerobacter ethanoli cus with simple chiral alcohols, (R)-(S)-2-butanol, and (R)- and (S)-2 -pentanol. HLADH does not show any effect of pH on enantiospecificity (E = R/S = 0.26) in the reaction of (R) and (S)-2-butanol, since ident ical pK(a)s of 8.0 are seen for both enantiomers. In contrast the reac tion of SADH with these alcohols shows significant influence of pH on the enantiospecificity. Ar 65 degrees C, the pK(a) for the reaction of SADH with 1-propanol, which is a poor substrate and hence has a low c ommitment, is 6.82 +/- 0.11. At 65 degrees C, the reaction of (R)-2-bu tanol exhibits a pK(a) of 6.60 +/- 0.10 while that of (S)-2-butanol ex hibits a pK(a) of 6.85 +/- 0.15. As a result, the E value for 2-butano l increases from 2.5 at pH 9 to 4.2 as pH 5.5. Similar effects are obs erved in the reaction of 2-pentanol with SADH. These data provide supp ort for the enantiomeric commitment model of pH-dependent enzymatic en antiospecificity. (C) 1996 by Elsevier Science Inc.