INHIBITION OF ENZYMES 825 



reversal was obtained in 59%, partial reversal in 30%, and no reversal in 

 11%. This shows at least that in most cases the enzyme is not seriously 

 altered or denatured by severe mercurial inhibition, providing the contact 

 with the inhibitor is not prolonged. On the other hand, there are the en- 

 zymes such as papain which are more stable when complexed with Hg++. 

 Some of the instances where partial or no reversal was found are undoubted- 

 ly due to inactivation, but some to the other factors mentioned above. In a 

 few studies, some interesting sidelights were noted and we shall content 

 ourselves with discussing some of these. 



Two more instances of very rapid, almost instantaneous reversal of mer- 

 curial inhibition may be noted. Yeast alcohol dehydrogenase is immediately 

 inhibited by p-MB and likewise rapidly reactivated by glutathione (Snod- 

 grass et al., 1960). The enzyme at 4.5 X 10"^ M is completely inhibited by 

 2.5 X 10"'' M Hg++; glutathione reverses this inhibition rapidly, but par- 

 tially — after 30-sec exposure to Hg++ 54% and after 10-min exposure 

 only 17%. This failure to reverse is not due to displaced Zn++, since Zn++ 

 was found not to be released from the enzyme so rapidly and the addition 

 of Zn++ does not improve the reversal. Heart lactate dehydrogenase is very 

 rapidly reactivated from p-MB inhibition by cysteine, and in this case the 

 rate of inhibition is relatively slow, about 15 min being required for maxi- 

 mal inhibition (Neilands, 1954). This is one of the few instances in which 

 reversal definitely seems to occur faster than inhibition. The effectiveness 

 of reversors in comparison with other methods for reactivation is seen with 

 succinate dehydrogenase. If the enzyme inhibited by p-MB is dialyzed for 

 3.5 hr there is no reactivation, but addition of glutathione reverses complete- 

 ly (Singer et al., 1956b). Also no reversal was observed following dilution of 

 the inhibited enzyme, whereas thiols reactivate partially (Slater, 1949). The 

 rate of reactivation of erythrocyte pyruvate kinase by 100 mM glutathione 

 after 1-min contact of the enzyme with 0.1 mM p-MB is fairly slow (see 

 accompanying tabulation); however, if contact with the inhibitor is 10 min, 



Time 

 (min) 



% Reversal 



10 18 



15 47 



25 59 



only 28% reversal is seen after 25-min exposure to glutathione (Solvonuk 

 and CoUier, 1955). There is no obvious explanation why the reversal rates 

 with some enzymes are so fast and in others so slow, especially as this is 

 not correlated at all with the aifinities of the enzymes for the mercurials. 



