SUBSTRATE INHIBITION 113 



A. The substrate in the active ES complex forms a multipoint attach- 

 ment to the enzyme (i.e., a substrate molecule is bound to the active cen- 

 ter by two or more enzyme groups) so at high substrate concentrations it 

 is possible that two or more substrate molecules can be bound simulta- 

 neously at the active center. If multipoint attachment is necessary for 

 reaction, binding of the substrate molecules at the single binding sites 

 will lead to inactive complexes and a reduction of available enzyme. 



B. The substrate may combine not only at the active substrate site but 

 also at sites in various spatial relationships to the active site, interfering 

 with either the binding of the substrate in an active ES complex or inter- 

 fering with its reaction when so bound. 



C. The substrate may react with an activator and at sufficiently high 

 concentration deplete the system of the activator so that the enzyme 

 becomes inactive. 



D. The true substrate for the enzyme may be a complex of the added 

 substrate with an activator, in which case the added substrate may com- 

 pete with this complex for binding to the enzyme. 



E. The substrate may interfere with the binding of a coenzyme or 

 acceptor in transfer reactions. 



F. The substrate at high concentrations may reduce the water concen- 

 tration and slow the rate if water is one of the reactants. 



G. Increasing substrate concentration may increase the ionic strength 

 of the reaction mixture and this may modify the rate independently of 

 any direct or specific effects of the substrate. 



Finally, it should be noted that reduction of rate at high substrate con- 

 centrations may be due not to the substrate itself but to the ions simulta- 

 neously added. If the substrate is an anion, for example, it is possible that 

 the sodium or potassium ions that accompany it may be responsible for 

 the inhibition, in which case the inhibition should not be interpreted as 

 true substrate inhibition. The kinetic behavior of the system in any case 

 will, of course, depend on the mechanism that is involved. 



The inhibition of dog liver p-hydroxyphenylpyruvate oxidase by excess 

 substrate (Zannoni and La Du, 1959) may be used to illustrate what may 

 be termed pseudosubstrate inhibition. Once the enzyme is inhibited by 

 high substrate concentrations it cannot be reactivated by prolonged dialy- 

 sis, but reducing agents such as 2,6-dichlorophenol-indophenol will bring 

 about a restoration of the activity. The evidence pointed to an inhibitory 

 substance formed from the substrate; the greater the substrate concen- 

 tration, the more of this substance was formed. Preliminary extraction 



