620 6. ARSENICALS 



(2) Failure to observe a potent inhibition by R — As=0 would not 

 prove the absence of a dithiol group because of various factors which might 

 impede the binding. It seems that in some respects arsenite is not always 

 the best test substance since usually the substituted arsenoxides inhibit 

 much more potently. The reason for this is not known but may involve 

 the small size of the arsenite, in that it will not interfere with the binding 

 of the substrate unless the SH groups are actually in the active site, whereas 

 larger R groups would be more likely to block sterically. It is also probable 

 that the arsenite is not as reactive with enzyme SH groups, since it usually 

 does not react as rapidly with simple thiols as do the substituted arse- 

 noxides. 



(3) Inhibition by R2=As — CI would not prove that there is only a 

 single SH group on the enzyme, since there is no apparent reason why 

 it would not react with one SH group of a pair just as well as with a single 

 SH group, as in reaction E. 



(4) Data from protection and reversal experiments often are completely 

 valueless for determining the nature of the reaction with the enzyme. 

 Just as protection or reversal by a thiol doesn't prove that an inhibitor 

 reacts with an enzyme SH group, protection or reversal by a dithiol doesn't 

 prove that an enzyme dithiol is involved. Such experiments only give some 

 indication of the relative affinities of the enzyme and the reverser for the 

 arsenical. Monothiols will generally be ineffective in preventing arsenical 

 inhibitions since in many cases stable complexes are not formed, and 

 dithiols, such as dimercaprol, will generally be effective whatever the 

 nature of the reaction with the enzyme. There are undoubtedly cases in 

 which an arsenical is bound to dimercaprol more strongly than to an enzyme, 

 and this may be true even though the enzyme has a dithiol group, so that 

 significant reactivation by dimercaprol certainly does not indicate whether 

 an enzyme is of the dithiol type or not. It must also be remembered that 

 some enzymes are inhibited by dimercaprol and this action may obscure 

 any reactivation produced, and that the degree of reactivation will depend 

 on whether the enzyme has undergone irreversible changes as a result of 

 arsenical binding. It is believed that only under exceptional circumstances 

 can protection or reversal data be used as evidence for the nature of enzyme 

 SH groups. 



(5) Occasionally the presence of a simple thiol markedly increases the 

 inhibition by an arsenical (see page 645). Thiol transacylase is not inhib- 

 ited at all by 5 mM arsenite, but in the presence of 2-mercaptoethanol the 

 enzyme is depressed 73% (Alberts et al., 1963). It was assumed, and prob- 

 ably rightly so, that these data do not indicate the enzyme to be of the 

 dithiol type. Succinic semialdehyde dehydrogenase is inhibited 50% by 

 4 milf arsenite, but with 2-mercaptoethanol it requires only 0.05 mM 



