INHIBITION OF ENZYM.ES 813 



groups are combined with mercurial (assuming that there is sufficient mer- 

 curial to react with all these groups). The more slowly developing inhibition 

 could be due to reaction of less readily available SH groups or to a secondary 

 inactivation following the initial mercaptide formation. Quite different re- 

 sults are obtained with amylase, mersalyl at 1 mM not inhibiting at all 

 during the first hour, but slowly inhibiting until there is 40% depression 

 after 48 hr (Muus et ah, 1956), or with bromelain, jJ-MB inhibiting only 

 25% after 4 hr and 80% after 20 hr at 0.1 mM (Ota et al, 1961). Many 

 different time courses of inhibition are observed and it is likely that the 

 major factors involved are (1) the relative reactivities of the SH groups, 



(2) the relationship between the SH groups and the catalytic activity, and 



(3) the tendency for structural changes leading to inactivation to occur. 

 However, it is quite clear that many enzymes react quite slowly with mer- 

 curials and require 2-4 hr (and occasionally more) to complete the process. 

 Such enzymes are difficult to titrate, since one does not know how many 

 of the SH groups finally reacted were originally present, and, when one is 

 adding increasing amounts of mercurial to correlate mercaptide formation 

 and inhibition, it is not easy to decide on the optimal preincubation in- 

 terval. 



In the previous section the correlation between inhibition and SH reac- 

 tion by mercurials was considered in terms of variable quantities of mer- 

 curial. Another approach to relate these phenomena is to determine their 

 changes with time at a particular mercurial concentration. If the SH groups 

 which are combined initially are necessary for enzyme activity, one would 

 expect inhibition to parallel blocking of these groups; if the most readily 

 reacting SH groups are not related to activity, or the enzyme undergoes 

 progressive inactivation, the inhibition may lag behind mercaptide forma- 

 tion. Madsen and Cori (1956) observed that inhibition of phosphorylase by 

 p-MB developed more slowly than the change in absorbance at 250 mjit 

 (Fig. 7-27), so that when 50% of the reactive SH groups had been blocked 

 the inhibition was only 14%. If the SH blocking itself is not responsible 

 directly for the inhibition, but initiates an unfolding of the enzyme, the 

 rate of inhibition may be more dependent on the rate of configurational 

 change. In the case of phosphorylase, we have seen that splitting into sub- 

 units occurs during reaction with p-MB, so the rate at which this occurs 

 may have something to do with the inhibition rate. Inasmuch as cysteine 

 reverses the inhibition completely, marked structural alterations would not 

 be very likely. 



Another phenomenon which must be taken into account in kinetic studies 

 is the spontaneous recovery of enzyme activity in the presence of the mer- 

 curial, first observed, I believe, by von Euler and Svanberg (1920) in studies 

 of the inhibition of yeast /5-fructofuranosidase by Hg++. Reisberg (1954) 

 reported that the inhibition of choline acetylase by p-MB is less at 30 min 



