146 4. SUBSTRATE INHIBITION AND PRODUCT INHIBITION 



high inhibitor concentrations, but were in the opposite direction from that 

 predicted for competitive effects, it was concluded that this simple treat- 

 ment was not completely adequate. However, this example does illustrate 

 that product inhibition can be complicated by deviations from the simple 

 formulation of Michaelis and Menten and that multiple substrates and sites 

 must be taken into account in elucidating specific mechanisms. 



The inhibition of cholinesterase by choline was first demonstrated by 

 Roepke (1937) in serum and the competitive nature of the inhibition indi- 

 cated by appropriate reciprocal plotting, the affinity for the choline being 

 comparable to that for acetylcholine which would be expected if the prin- 

 cipal binding energy is derived from the anionic site attachment. Similar 

 results were obtained by Ziff et al. (1938) using a different procedure for 

 the evaluation of the kinetics and subsequent work has confirmed this 

 picture of product inhibition for the serum enzyme. A more thorough 

 investigation was made by Augustinsson (1948) and completely compe- 

 titive inhibition was shown for horse serum cholinesterase using a variety 

 of substrates. However, for the erythrocyte and brain enzymes the curves 

 deviated from linearity, although a competitive type of inhibition was 

 suggested by the fact that the curves all passed through a single point on 

 the ordinate. The explanation is that for these tissue cholinesterases we 

 have both substrate and product inhibition simultaneously. The presence 

 of choline shifts the substrate concentration for maximal rate (Sq) to higher 

 values as would any competitive inhibitor; a concentration of choline nec- 

 essary for 44% inhibition increased (Sq) about ten-fold. It is interesting 

 that the cholinesterase from Helix dart sac, which does not exhibit marked 

 substrate inhibition, gave linear curves in the usual l/v — 1/(S) plot but 

 these curves did not intersect on the l/v axis nor on the 1/(S) axis. It is 

 possible that this indicates mixed inhibition where the binding of choline 

 does not completely prevent binding of acetylcholine; extrapolating the 

 curves to their intersection indicates that choline increases the Kg of ace- 

 tylcholine some seven-fold (see Chapter 5). 



The noncompetitive nature of much product inhibition has been sur- 

 prising to some investigators because the usual structural similarity be- 

 tween product and substrate implies reaction at the same site. However, 

 if it is assumed that there are two sites, a substrate site forming ES and 

 a product site forming EP, and that ES is transformed into EP in the course 

 of the reaction, the following scheme represents the situation: 



S 

 E 



E 



\ 



/l"V /^ 



X ^/ P 



E (4-46) 



\ 



E 



