Although there has been a great deal of interest in the effects of physical 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 theoretical 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 enzymatic stereochemistry based on differential catalytic commitments for enantiomeric substrates. This model predicts that enantiomeric substrates 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 on the reactions of both horse liver alcohol dehydrogenase (HLADH) and secondary alcohol dehydrogenase (SADH) from Thermoanerobacter ethanolicus 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 identical pK(a)s of 8.0 are seen for both enantiomers. In contrast the reaction 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 commitment, is 6.82 +/- 0.11. At 65 degrees C, the reaction of (R)-2-butanol exhibits a pK(a) of 6.60 +/- 0.10 while that of (S)-2-butanol exhibits a pK(a) of 6.85 +/- 0.15. As a result, the E value for 2-butanol increases from 2.5 at pH 9 to 4.2 as pH 5.5. Similar effects are observed in the reaction of 2-pentanol with SADH. These data provide support for the enantiomeric commitment model of pH-dependent enzymatic enantiospecificity.