810 15. EFFECTS OF VAEIOUS FACTORS ON INHIBITION 



Since K^,^ in this case is kj^i (k-i being negligible compared to k^), it 

 was concluded that /-^ must decrease as the dielectric constant is lowered. 

 The possibility that methanol was acting as a competitive inhibitor was 

 eliminated. The formation of the ES complex is thus slowed by a reduction 

 of the dielectric constant and this would mean that either the substrate 

 and the active site have ionic charges of the same sign or a dipolar inter- 

 action involving H+ or 0H~ transfer from the solution occurs. Since the 

 substrate is not ionic, it is likely that the second explanation is correct. 

 However, the possibility of effects of the dielectric constant on the ioniza- 

 tion of the imidazole group at the active center should also be considered. 



Laidler has used these data and his own to determine the entropies of 

 activation for the formation of the ES complex and for its subsequent reac- 

 tion (Laidler, 1958, p. 206; Barnard and Laidler, 1952). Liasmuch as the' 

 variation of the free energy of activation with the temperature allows one 

 to calculate the activation entropy, and since the free energy can be par- 

 titioned into electrostatic and nonelectrostatic fractions, it is possible to 

 determine the contributions of these two fractions to the over-all entropy 

 change. These activation entropies are very useful in interpreting the mo- 

 lecular events occurring during the enzyme reaction — changes in hydra- 

 tion or the state of water, separation of charges, structural modification of 

 the enzyme protein, and related events — but Laidler has pointed out 

 the difficulties involved in work with mixed solvents and one really cannot 

 be at all certain that these deductions are quantitatively valid. 



A very interesting piece of work — and a disturbing one to lovers of 

 simplicity — on the effects of organic solvents on the hydrolysis of methyl 

 hippurate by a-chymotrypsin has been reported more recently by Apple- 

 white and associates (1958) and the results cast some doubt on the in- 

 terpretation of the rate effects as due entirely to changes in the dielectric 

 constant. A number of organic substances were used and the effects on 

 the kinetic constants often differed (Table 15-2). For example, methanol 

 increases K^.^ and decreases A'2, whereas acetone increases K,,, but has no 

 effect on k^. A general conclusion is that in all cases there is a rise in the 

 Michaelis constant, K„j, and this probably reflects the response of k-^ to 

 the lower dielectric constant. Furthermore, since ko is unchanged by metha- 

 nol when acetyl-L-tyrosinamide is the substrate (Kaufman and Neurath, 

 1949) and is markedly decreased when methyl hippurate is the substrate, 

 different reaction sequences must be postulated. Finally, plots of log ^,„ 

 against 1/Z), or l/D-, or {D — l)l(2D + 1), are all nonlinear. There is a 

 rough correlation between TiT,,, and the dielectric constant in those cases 

 where A^g is unchanged but it is not sufficient to conclude that the dielectric 

 constant is the sole factor involved. 



(6) Myosin adenosinetriphosphatase. Inasmuch as ATP is a highly charged 

 anionic substrate, it would be reasonable to expect a large electrostatic 



