692 



14. EFFECTS OF pH ON ENZYME INHIBITION 



inhibition when the carboxyl groups on the inhibitors are esterified or 

 replaced by nonpolar groups, combined with the retention of the inhibi- 

 tion if the carboxyl group is replaced by a sulfonic acid group. Inasmuch 

 as the range of pH covered was from 5.7 to 9, the inflections that would 

 indicate the ionizations of the inhibitor carboxyl groups are not seen. 



coo- 



coo- 



coo- 



coo- 



The curves for maleate, D-tartrate, mesaconate, and succinate seem to 

 be of a configuration indicating either the active form of the enzyme to 

 be HgE or the active form of the inhibitor to be Hoi. Since it is unlikely 

 that the inhibitors would be active in the uncharged form, this would 

 point to two grouj)s on the enzyme that must be protonated in order to 

 combine with these anions, one with a itK^ of 6.5 or below, the other near 

 7.5-8.0. Results with other inhibitors and with the substrates, fumarate 

 and L-malate, also point to enzyme groups with p^^'s of similar magnitude. 

 The p^,^ of approximately 6.5 might correspond to an imidazole group 

 in histidine at the active center while the pK^ near 8 could represent an 

 a-amino group (or possibly a phenolic group in tyrosine); these two groups 

 would serve to bind the substrates and inhibitors to the enzyme, and in 

 the former case to participate in the transfer of hydrogen atoms. Sulfhy- 

 dryl groups are excluded because of the insensitivity of the enzyme to 

 reagents reacting with these groups (iodoacetate, iodoacetamide, iodoso- 

 benzoate, p-chloromercuribenzoate, and chloroacetophenone). 



It is interesting that the ^raws-aconitate curve shows no inflections at 

 all over the pH range studied. Since there is good evidence that there are 

 ionizing groups on the enzyme, it is remarkable that no alteration of the 

 curve is observed at pH's corresponding to the pKJs of these groups, and 

 the behavior must be considered, for the time being, as anomalous. The in- 



