VARIATION OF MULTIENZYME INHIBITION WITH pH 711 



D. The determination of K^ values at different pH's should be done with 

 different methods of plotting so that the relationship between K- and K^ 

 can be more certainly established. 



E. In comparing inhibitors on an enzyme, take the pH effect into account 

 and determine the true inhibitor constant, K^, since it is only from K^ 

 that the relative affinities and binding energies may be calculated. 



F. It is better to determine directly the pH of each enzyme reaction me- 

 dium, after all additions and at the beginning and end of the experimental 

 interval, rather than trust a buffer throughout the range of pH covered. 



G. In all studies of enzymes, including inhibition, use the lowest con- 

 centration of buffer possible under the circumstances so as to avoid buffer 

 effects that may complicate the kinetics. Most buffers are substances that 

 do not occur within cells, or not at the concentrations commonly used, and 

 as a general principle it is advisable to introduce as few unnatu-ral substan- 

 ces as possible and to use media that are as close in composition to the cy- 

 toplasm as our information allows. 



VARIATION OF MULTIENZYME INHIBITION WITH pH 



The kinetics of multienzyme systems were found in Chapter 7 to be 

 fairly complex and the addition of i^H dependence for each of the steps, due 

 to ionization of substrates, enzymes, or both, allows the kinetics to be ex- 

 tended in a straightforward manner but often makes the interpretation of 

 experimental results difficult unless the behavior of each enzyme in the 

 system is understood. Yet it is with such problems that one must deal in 

 investigating the response of particulate or cellular metabolic systems to 

 changes in the pH. The individual rates of the various reactions in a multi- 

 enzyme system will generally show different types of dependence on the 

 pH and thus any variation in the pH will alter the steady-state character- 

 istics and the over-all flow pattern. If the substrate. A, and the interme- 

 diate, B, in the simple monolinear chain, A ^ B -> C, both ionize, the con- 

 centration of the active form of B will change with pH not only because 

 of the direct effect on its ionization but also because of the altered rates 

 of the two reactions. In a divergent chain, as in scheme 7-25, the relative 

 rates at which C and D are formed may be dependent on the pH, since 

 Eg and Eg will be affected differently by changes in the pH. It is on the 

 background of such behavior that inhibition in these systems must be 

 considered. 



The steady-state kinetics for multienzyme systems are usually obtained 

 by expressing the rates of the various reactions — v-^^, i\, v^, ... — and then 

 relating these rates appropriately in an equation. When the pH is to be 



