814 15. EFFECTS OF VARIOUS FACTORS ON INHIBITION 



partially reported. Plots of (S)/v against (S) at different concentrations of 

 methanol show that the slope is changed but the intercept remains con- 

 stant. This means that k^ is decreased with increasing methanol or decreas- 

 ing dielectric constant and that ^,„ is relatively unaffected. Since ^,„ 

 for carboxypeptidase is probably k^jk-y, this would indicate that k^ must 

 also decrease in media of lower dielectric constant. However, the other 

 organic substances (ethanol, isopropanol, and dioxane) had pronounced 

 effects on the intercept indicating a marked increase in k^. It was iDointed 

 out that these results with other solvents make the interpretation of the 

 methanol work more difficult, but might point to electrostatic rearrange- 

 ments in the activated complex of the rate-limiting step. 



(e) Yeast enolase. Methanol, ethanol, and dioxane all depress the rate of 

 the enolase reaction under conditions in which the enzyme is saturated 

 with Mg++ and the substrate (Westhead and Malmstrom, 1957). When the 

 rate is plotted against the concentration of the organic solvent, the points 

 all fall essentially on one curve, although the dielectric constant changes 

 produced by these substances are quite different. The decreased rate must 

 be due to either a decrease of A-g or of the concentration of active enzyme. 

 Temperature studies showed there is no effect on the activation energies 

 and thus it is unlikely that k^ is altered. Denaturation of the enzyme by 

 the solvents or the formation of inactive aggregates by association were 

 eliminated as possible explanations. It was concluded that the solvents 

 acted by displacement of water rather than by a dielectric effect. This de- 

 crease in the water concentration might affect the degree of dissociation of 

 proton-transferring groups at the active center. If this explanation is cor- 

 rect, it would emphasize the importance of considering the effects of or- 

 ganic solvents on ionization constants and proton association, factors sel- 

 dom mentioned in work on other enzymes. 



For the useful interpretation of studies in which the dielectric constant 

 is changed, the attitude expressed by Westhead and Malmstrom is neces- 

 sary. "While useful information may be obtained from the use of solvent 

 mixtures in studying enzyme kinetics, the situation may be very complex. 

 Results obtained from limited experiments may easily be subject to gross 

 errors in interpretation," This point is particularly important and one is 

 justified in being sceptical of conclusions based on the use of only one 

 organic solvent. Ideally such work should include results from several 

 solvents over as wide a range of concentrations as possible and should 

 test the effects of these solvents on the pH and temperature characteristics 

 of the reaction studied. Furthermore, all the possibilities by which these 

 solvents may alter the kinetics must be examined and the interpretation 

 must not be based solely on the changes in the dielectric constant. 



The effects of solvent-water mixtures on enzyme kinetics have been 

 discussed in some detail because there have been no studies on inhibition. 



