INHIBITION OF ENZYMES 785 



structure rather than bind the NADH; disintegration of the structure 

 brought about by p-MB would secondarily lead to release of the coenzyme. 

 As the NADH is split from the apoenzyme by p-MB, rotatory dispersion 

 titration indicates changes in optical rotation associated with denaturation, 

 so that Li et al. (1962) likewise inclined to a theory involving structural 

 changes as a basis for the displacement, since it is known that denaturation 

 by heat or other agents releases the NADH. Yonetani and Theorell (1962) 

 have used the very sensitive spectrofluorometric method for the measure- 

 ment of NADH binding and dissociation.* They demonstrated that the en- 

 zyme configuration is stabilized by the NADH and additionally by the iso- 

 butyramide, although these can be associated directly with only a small 

 fraction of the total number of SH groups, and suggested that denaturation 

 may be Initiated by local changes at the active centers and from there 

 spread throughout the molecule. The SH groups may form a network of 

 hydrogen bonds contributing to the stability of the tertiary structure, so 

 that mercurials could create instability either locally or generally. All of 

 this recent work shows that mercurials probably induce configurational 

 changes in the enzyme, these being irreversible by the usual means, and 

 they provide an alternative explanation for NADH release, but do not dis- 

 prove the original hypothesis that a direct binding between NADH and 

 SH groups occurs. The Zn++-dependent alcohol dehydrogenase of yeast is 

 inhibited by mercurials, and this was attributed to displacement of the 

 Zn++ from SH groups (Wallenfels and Sund, 1957 a) on the basis that res- 

 toration of activity requires both glutathione and Zn++. However, an inves- 

 tigation of the time course of the inhibition showed that glutathione alone 

 is sufficient to reactivate if it is added soon after the mercurial, but the inhi- 

 bition progressively becomes irreversible, at which time no Zn++ has been 

 released (Snodgrass and Hoch, 1959). Zn++ is displaced progressively over 

 a period of several hours, but the inhibition does not appear to be mediat- 

 ed through this displacement. Such a slow release, without correlation with 

 the mercurial reaction or the inhibition, is probably due to structural 

 changes in the enzyme. 



The early work on NADH splitting from alcohol dehydrogenase was soon 

 confirmed for 3-phosphoglyceraldehyde dehydrogenase (3-PGDH) by Velick 

 (1953). The yeast enzyme requires 2 SH groups for full activity and inhi- 

 bition by p-MPS increases until 2 equivalents of the mercurial are added. 

 In contrast to the alcohol dehydrogenase, this inhibition is readily revers- 



* Liver alcohol dehydrogenase forms a very stable ternary complex with NADH 

 and t«obutyramide, and this complex is strongly fluorescent. The enzyme may be 

 titrated in the presence of 100 mM isobutyramide with NADH, measuring the fluo- 

 rescence increase at 410 m/<, and then back-titrated with p-MB or p-MPS as the NADH 

 is released from the apoenzyme. This will probably be a very valuable technique for 

 the study of coenzyme binding. 



