INHIBITION OF ENZYMES 827 



Unusual results on the effects of thiols on the inhibition of /?-fructofu- 

 ranosidase by Hg++ were observed by Gemmill and Bowman (1960), in 

 that dimercaprol reverses the inhibition to varying degrees, whereas both 

 cysteine and glutathione increase the inhibition (Table 7-12). The thiols 

 themselves, even at the highest concentrations used, do not significantly 

 affect the enzyme activity, so the additional inhibition cannot be attribut- 

 ed to an excess of the thiol. Several explanations are possible: (1) The cys- 

 teine and glutathione reduce enzyme disulfide groups to SH groups and 

 increase the binding of the Hg++ to the enzyme; (2) the Hg++ reacts with 

 both enzyme SH groups and these thiols to form E — S — Hg — S — R com- 

 plexes which are less active than the simple E — S — Hg+ complexes; or (3) 

 the R — S — HgX or R — S — Hg — S — R mercaptides formed are inhibitory 

 by a mechanism unrelated to enzyme SH groups. Whatever the explanation 

 applicable here, one would expect that these situations would occasionally 

 be important in the reversal of other enzyme inhibitions, particularly when 

 the bifunctional Hg++ is used, and possibly failure to achieve reversal in 

 some cases may be due to these reactions. 



Complexities arise in some reversal studies and a few will be mentioned 

 briefly. When the aspartate: or-ketoglutarate transaminase from pig heart 

 is treated with p-MB there is inactivation but the pyridoxal-P remains 

 bound to the apoenzyme (Turano et al., 1964). Incubation for 10 hr at pH 

 6.4 and 4° with 0.35 mM p-MB, however, releases the coenzyme. The ad- 

 dition of pyridoxal-P restores the activity in the sense that addition of 

 glutamate is followed by transamination to form pyridoxamine-P, but the 

 further transamination to oxalacetate is lost. In this instance, reversal thus 

 occurs for only part of the normal reaction. Carboxy peptidase A is activat- 

 ed by Zn++ and apparently the Zn++ is bound to the SH groups of a single 

 cysteine residue and the a-amino group of a terminal asparagine (Coombs 

 et al., 1964). Zn++ protects the enzyme from inhibition by p-MB, but if the 

 apocarboxypeptidase is inactivated by p-MB, addition of Zn++ does not 

 restore the activity. 



Spontaneous reversal of enzyme inhibition by p-MB occurs with 3-phos- 

 phoglyceraldehyde dehydrogenase of muscle (Szabolcsi et al., 1960), and 

 has occasionally been observed in other systems, particularly homogenates. 

 This has usually been attributed to redistribution of the mercurial from the 

 rapidly reacting SH groups to others which bind the mercurials more tight- 

 ly, but in this case it was thought that intermolecular rearrangements, 

 whereby eventually some of the enzyme is inactive and some completely 

 active, are responsible. This should not be too uncommon a phenomenon 

 if the proper mercurial concentrations are used. 



