836 



15. EFFECTS OF VARIOUS FACTORS ON INHIBITION 



strength effects was made by Myers (1950) using cholinesterase from human 

 erythrocytes and serum. The inhibitions by physostigmine and neostigmine 

 on both enzymes are reduced by adding NaCl to the usual bicarbonate me- 

 dia. On the average, the pljo is decreased by around 0.5-0.6 when the ionic 

 strength is increased by approximately 0.16. A concentration of 5x 10"^ M 

 inhibits the erythrocyte enzyme 61% in the bicarbonate medium and 28% 

 when 0.125 M NaCl is added. KCl produces the same effect and CaClg 

 is more effective. The accompanying tabulation shows the effects of various 



salts on the physostigmine inhibition of erythrocyte cholinesterase. An 

 increase in the ionic strength thus reduces the affinity of the enzymes 

 for the inhibitors, just as it reduces the affinity for acetylcholine, and 

 this is reasonable on the basis of a positively charged inhibitor interacting 

 with a negatively charged group on the active site. The inhibition pro- 

 duced by cliisopropylfluorophosphate, on the other hand, is unaffected 

 by the ionic strength. 



Proteolytic enzymes have been studied rather extensively. Phenyl- 

 acetate and 3-indolepropionate are competitive inhibitors of chymotrypsin 

 and they are more effective when the ionic strength is increased (Bernhard 

 and Niemann, 1957). The K^ for the latter inhibitor is 7.6 mM in the ab- 

 sence of phosphate and is not changed as the phosphate concentration is 

 increased to 10 mM , but then begins to decrease until it is 2.5 at a phosphate 

 concentration of 400 mM {s = 1.09). These inhibitors are negatively 

 charged and it is postulated that a negative group is situated near the active 

 center. An increase in the ionic strength would reduce the repulsion and 

 increase the affinity of the enzyme for the inhibitors. There is evidence 

 that there is also a specific ion effect of phosphate. Certain polyanions such 

 as poly-a-L-glutamate, polycysteate, and heparin, inhibit trypsin and it 

 appears that this is due to a complex formed with the enzyme (Kornguth 

 and Stahmann, 1960). It is postulated that they combine with positively 

 charged groups on either side of the active site, forming a bridge over the 

 site to block off the substrate. The inhibition is markedly decreased by an 

 increase in the ionic strength (Table 15-6) and this supports the electro- 

 static nature of the binding. It may be noted that the smaller inhibitions 



