382 7. INHIBITION IN MULTIENZYME SYSTEMS 



not difficult if the enzymes can be isolated or studied independently and 

 they would provide a better understanding of many multienzyme systems. 



When more complex multienzyme systems are considered, it is clear 

 that the concept of limiting rate is even more vague and often inapplicable. 

 In cyclic or regenerative systems, although one step may be said to be more 

 important in controlling the rate than any other step, no one step can be 

 the sole determinant, as indicated by the fact that inhibition on any step 

 will depress the over-all rate to some extent. Furthermore, occasionally the 

 controlling role must be attributed to more than one reaction. For example, 

 under certain circumstances it is believed that the triose-P dehydrogenase 

 step is the limiting reaction in glycolysis. Since this may be so because 

 of a low concentration of ADP, the formation of pyruvate of lactate may be 

 said to be limited by some reaction forming ADP from ATP; inhibition of 

 either the triose-P dehydrogenase or the ATPase will depress glycolysis. 

 In very complex metabolic systems there may be several reactions sensi- 

 tive to inhibition with respect to the rate of formation of some product. 



The danger of assuming a certain reaction step to be limiting a complex 

 metabolic process is illustrated in the fine study of metabolic regulatory 

 mechanisms by Wu (1959). Although the rate of lactate formation in HeLa 

 cells was the same under either aerobic or anaerobic conditions, the lactate 

 formed from endogenous carbohydrate showed a pronounced Pasteur ef- 

 fect. It was demonstrated that the rate-limiting step in endogenous gly- 

 cogenolysis is glucosan phosphorylase. On the other hand, the conversion 

 of glucose to lactate in cells grown in horse serum appears to be limited by 

 phosphofructokinase, whereas in cells grown in human serum, the limiting 

 factor is the inorganic phosphate entering into the phosphoglyceraldehyde 

 dehydrogenase reaction. Thus the same cells can show at least three differ- 

 ent rate-limiting steps under various conditions. Such results make the 

 assignment of limiting steps in the cells or tissue being studied, based only 

 on work with other types of cells or different species, completely unreliable. 



Within the cell it is probable that the evolution of metabolic pathways 

 has resulted in systems in which the component reactions are balanced so 

 that no one step is predominantly limiting, at least under all conditions. 

 The various processes controlling metabolic rates, such as adaptive enzyme 

 formation, also tend to lessen the occurrence of markedly limiting reactions. 

 In any event, one is on safer ground in assuming the absence of limiting 

 reactions until they are proved to occur, and even then it is well to remem- 

 ber that a limiting state may exist only within a certain range of conditions. 



Determination of Substrate and Inhibitor Constants 



Determination of the constants, K,,^ and Ti,, by reciprocal plotting or 

 related methods will often not be possible in multienzyme systems. The 

 variation in rate with either (S) or (I) has been shown above to deviate 



