876 16. SPECIFICITY OF INHIBITION 



and the inhibitions on Eg and E3 are minimal. Setting %2 =^ ^23 it is found 

 that the inhibitor concentrations must be: 



'11 '23 h2 '21 



Substituting the numerical values for the inhibitor constants: (I^) = 0.20 

 mM; (I2) = 0.33 mM; i^^^ = 0.50; i^^ = 0.0625; i^^ = 0.0625. If the inhi- 

 bitors are used singly to inhibit E^ by 50%, (I^) must be 0.30 mM and (Ig) 

 must be 1 mM, the inhibitions on E2 and E3 being 9.1% and 16.7% res- 

 pectively, so that the combination has improved the specificity signi- 

 ficantly. 



If it is now desired that the inhibitions on Eg and E3 do not exceed 

 10% and the inhibition on E^ be maximal, we may set (I^) = 0.11 K^ 

 and (I2) =0.11 K,^^. In the example chosen, (I^) = 0.33 mM and (Ig) = 

 0.55 mM, so that this combination of inhibitors would inhibit E^ by 62.5%. 

 To achieve this degree of inhibition on E^ with the inhibitors singly, the 

 inhibitions on the other enzymes would be much higher than 10%. 



Finally, if maximal specificity is desired, independent of the absolute 

 values of the individual inhibitions, reference to Table 16-2 shows that the 

 maximal specificity between E^ and Eg, 0^2. = 0.52, and between E^ and 

 E3, o'j3 = 0.38, when the inhibitors are used singly. However, combinations 

 can increase both specificities, a-^^ fo as much as 0.61 and 0-13 to as much as 

 0.69, but not simultaneously. If the maximal specificity with respect to 

 Eg is desired, some sacrifice in the specificity towards E3 must be made, 

 and vice versa. The actual combination chosen will depend on the nature 

 of the work and the relative importance of the subsidiary enzymes in the 

 system being studied. 



This approach may be readily extended to combinations of three or more 

 inhibitors and even greater specificity may often be achieved. It may also 

 be applied to inhibitions on multienzyme systems or metabolic sequences, 

 but here it is difficult to express the relationships quantitatively and 

 predict the optimal inhibitor concentrations. For example, if one wished 

 to inhibit the glycolytic pathway with minimal inhibition on the tricarbox- 

 ylic acid cycle, it might be possible to achieve greater specificity with a 

 combination of iodoacetate, fluoride, and a glucose analog than with any 

 single inhibitor, but the determination of the proper concentrations to use 

 would depend on accurate experimental data using the inhibitors alone. 

 It is worth repeating that in studies attempting to correlate some cellular 

 function with a particular enzyme or metabolic process, it is generally 



