492 10. EFFECTS OF MORE THAN ONE INHIBITOR 



in which I^Ia is not formed will depend on the relative inhibitory potency 

 of Ijl., compared to the uncomplexed inhibitors, and the amount of the 

 change in inhil)ition will depend primarily on K^. Thus the reaction between 

 two inhibitors can result in either antagonism or potentiation of the inhi- 

 bition. If one of the inhibitors is a substance present in the enzyme test 

 medium, its presence will distort the response to the added inhibitor and 

 produce nonlinearity in the usual plotting procedures. 



Example of Positive Interaction: Inhibition of Succinic Oxidase by Fluoride 



and Phosphate 



A very interesting instance of an unexpectedly potent effect produced 

 by a combination of two inhibitors was reported by Slater and Bonner 

 (1952). Both phosphate and fluoride were found to be rather weak compet- 

 itive inhibitors of cardiac succinic dehydrogenase but in the presence of 

 the two ions the enzyme was depressed much more than would be predicted 

 by the individual inhibitions. The kinetics here are different than those 

 expressed in Eqs. 10-3 and 10-4 for competitive inhibition because both 

 inhibitors can be bound to the enzyme simultaneously and a no longer is 

 infinity. We must now write: 



'"' ^ (S)' + 1 + (P)' + (F)' + [(P)'(F)7a] 



where (S)', (P)', and (F)' are the specific concentrations of succinate, phos- 

 phate, and fluoride respectively. Instead of determining the total inhibition 

 produced by phosphate and fluoride acting together, let us rather consider 

 the inhibition by fluoride in various concentrations of phosphate. This will 

 be expressed by if and is given by: 



(10-16) 



The constants determined liy Slater and Bonner are: K,,^ = 0.48 mM 

 (it has been shown for succinic dehydrogenase that K^,^ is not the substrate 

 dissociation constant), K^ = 100 mM and Kf = 20 mM. They also found 

 that when phosphate is bound to the enzyme, the dissociation constant 

 for fluoride is reduced from 20 mM to 0.067 mM, so that a = 0.00335, 

 indicating a remarkable and unexplained positive interaction between the 

 inhibitors. When these constants are substituted into Eq. 10-16 the in- 

 hibition curves for fluoride shown in Fig. 10-1 are obtained. The marked 

 potentiation is evident: in the absence of phosphate, 10 mM fluoride in- 

 hibits 2.2% whereas when the phosphate is 50 mM (which in itself would 

 produce only 2.2% inhibition), the inhibition by fluoride would be 77%. 



