FACTORS IMPORTANT IN INHIBITION 99 



reactions of the inhibitor. This means that the concentration of active 

 enzyme (E^) is decreasing during the experimental interval, often resulting 

 in significant deviation from inhibition kinetics in systems where the 

 enzyme is stable. Certain isolated enzymes are unstable under the condi- 

 tions for the determination of enzyme rate since they are operating in an 

 environment abnormal compared to their intracellular state. Many en- 

 zymes may be reasonably stable at low temperatures but are inactivated at 

 a rather rapid rate at 37-38" when isolated. Inhibitors, especially those 

 reacting chemically with protein groups, may induce a characteristic inhi- 

 bition at or near the active center but in addition may alter the protein 

 structure, or denature, which will secondarily affect the enzymic activity. 

 If the decrease in active enzyme concentration is linear, or approximately 

 so over the experimental interval, there will be no effect on the inhibition 

 measured for this interval if the enzyme inactivation is spontaneous and 

 occurs at the same rate in the presence and absence of the inhibitor, since 

 both uninhibited and inhibited rates will be similarly affected, but if the 

 inactivation occurs only in the presence of the inhibitor, the inhibition 

 is changed according to the following equation: 



• (D + 1/2 [1 - {Vm'lVJ] .„ ,... 



^ = ^p^^p^^ (3- 102) 



where F„; is the initial maximal rate and F,,/ is the maximal rate at 

 the end of the experimental period. F,„ and F„/ are, of course, proportio- 

 nal to the concentrations of active enzyme at the two times. Thus a non- 

 competitive inhibition of 50% by (F) would be raised to 62.5% if half the 

 enzyme were inactivated during the experimental interval. Determination 

 of K^ may be incorrect under such circumstances. Indeed, (I)o.5 = (Vy/ 1 

 V^)K^ so that the determined K^ may be in error by the ratio of final to 

 initial active enzyme concentration. If the decrease in (E^) is nonlinear, the 

 expression for the over-all inhibition is more, complex; however, if the inac- 

 tivation is first-order or logarithmic, it is clear that the effect on the inhi- 

 bition will be even greater than for the linear inactivation. 



It is well to bear such effects in mind in much inhibition study but it 

 must be admitted that it is usually difficult to demonstrate such secondary 

 inactivation if it is due to the enzyme, much less plot its time course. 

 Ideally one would like to be able to determine the concentration of active 

 enzyme at the termination of the experimental interval, but it is not possi- 

 ble to remove the inhibitor from the enzyme immediately, especially those 

 inhibitors likely to produce secondary inactivation. However, if Ijv^ is 

 plotted against (I) (see Chapter 5), the true K^ can be determined; substi- 

 tution of this value into the usual inhibition equation (enzyme is stable) 

 will demonstrate any deviation and enable Vy,/ IV„^ to be calculated approx- 

 imately. Or the inhibitor concentration corresponding to the determined 



