INHIBITION OF ENZYMES 49 



will be restored. Finally, if a portion of the inhibition is due to iodine in 

 the iodoacetate, thiol would be likely to reverse this. 



Alkylation of Certain Enzymes in the Glycolytic Pathway 



Iodoacetate and related alkylating agents have recently been used to 

 characterize the active sites of enzymes. For this purpose quite high con- 

 centrations are often used so that these studies provide no information on 

 effects obtained in cellular preparations, but the results in themselves are 

 quite interesting and illustrate the value of these substances in the delinea- 

 tion of active sites. When yeast enolase is incubated with 300 xnM bromo- 

 acetate for 2 hr at pH 7 and 35o, there is 90% inactivation and the car- 

 boxymethylation of "2.7 histidine residues, 3 methionine residues, and 1 ly- 

 sine residue (Brake and Wold, 1962). If 2-phosphoglycerate and Mg++ are 

 present, only 1.6 methionine residues are attacked and the enzyme activity 

 is less reduced. Thus it appears that the inhibition of enolase by bromo- 

 acetate is due to the carboxymethylation of 1 or 2 methionine residues at 

 the active site. 



The SH groups of phosphoglucomutase treated with 67 mM iodoaceta- 

 mide for 10 min at pH 8 and 25^ are not carbamylmethylated, but in the 

 presence of the substrate one SH group becomes more reactive (Koshland 

 et al., 1962). This picture was modified somewhat by using longer incubation 

 periods of 180 min and 270 mM iodoacetamide, from which it is seen that 

 in the first 10 min the substrate accelerates alkylation but after that it 

 protects, although incompletely (Koshland, 1964). It was questioned wheth- 

 er the protection observed is due to the presence of cysteine, methionine, 

 and lysine at the active site, or to changes in the configuration of the en- 

 zyme brought about by glucose-6-P. 



The results obtained on aldolase from rabbit muscle are puzzling. If the 

 enzyme is first incubated with /?-mercaptoethanol to ensure reduction of all 

 available disulfide groups, the thiol then removed, and incubated with iodo- 

 acetamide in urea for 2 hr at pH 7.5 and 40°, no free SH groups are detect- 

 able (Westhead et al, 1963). If now the enzyme is retreated with /?-mer- 

 captoethanol, one SH group appears. This could be explained either by 

 assuming that the aldolase is not homogeneous, essentially half the enzyme 

 containing a disulfide group and half not, or by visualizing a lone SH group 

 existing in some combined form other than disulfide. 



Kinetics of Inhibition 



Iodoacetate and iodoacetamide usually inactivate enzymes slowly, and 

 sometimes very slowly, as has been known since the work of Hopkins and 

 Morgan (1938) on succinate dehydrogenase, at which time they showed 



