48 1. lODOACETATE AND lODOACETAMIDE 



the enzyme against secondary inactivation as it often can against enzyme 

 denaturation. Furthermore, faihire of a substance to protect does not nec- 

 essarily prove that the SH group is unassociated with the binding location 

 of the substance but might be due to (a) too low a relative affinity for the 

 enzyme, (b) too low a concentration used, (c) inability to cover adequately 

 the particular SH group, or (d) too long an incubation period (since a pro- 

 tector generally slows the rate of inhibition but does not change the final 

 degree of inhibition). 



Reversibility of lodoacetate Inhibition 



If the inhibition arises from carboxymethylation of an enzyme group, 

 one would expect the inhibition to be very slowly reversible or completely 

 irreversible upon removal of the iodoacetate. Most of the early workers 

 reported irreversibility (Hopkins et al., 1938; Barron and Levine, 1952), 

 but there have been a surprisingly large number of claims for reversibility. 

 For exam])le, Walsh and Walsh (1948) found fructose- 1,6-diphosphatase, 

 inhibited 40% by 10 mM iodoacetate, to be completely reactivated by 

 cysteine, and Rocca and Ghiretti (1958) state that cysteine will completely 

 reverse the inhibition of D-glutamate oxidase. In some instances dialysis is 

 sufficient to reactivate, as with arylsulfatase (Maengwyn-Davies and Fried- 

 enwald, 1954) and papain (Weissmann et al., 1960). It is somewhat unex- 

 pected that Fe++ reactivates the enzyme responsible for the reaction 3- 

 hydroxyanthranilate — > quinolinate (Miyake et al., 1954). 



Various possibilities for explaining such reactivation come to mind. It is 

 quite possible that in some cases a hydrolysis of the carboxymethylcysteine 

 bond occurs: 



E— S— CH^— COO- + H^O -> E— SH + HO— CH^COO" 

 particularly if the pH is favorable, or possibly a thiol can react as follows: 



E— S— CH2— COO- + HS— R -> E— S— S— R + H3C— COO- 

 E— S— S— R + HS— R -> E— SH + R— S— S— R 



Maengwyn-Davies and Friedenwald (1954) thought that, in the case of 

 arylsulfatase, the iodoacetate (which was 20 mM) might have reacted with 

 the product of the reaction, ?)-nitrophenol, instead of on the enzyme. A 

 more likely general explanation is that a fraction of the enzyme SH groups 

 are in the oxidized disulfide form and cannot be carboxymethylated. Let 

 us assume that half the SH groups are in the oxidized state and the enzyme 

 half active; addition of a thiol activates the enzyme fully. If iodoacetate 

 reacts completely with the free SH groups and not with the disulfide groups, 

 the enzyme will be inhibited 100%; if a thiol is now added, the activity 



