INHIBITION OF ENZYMES 617 



Cysteine ^^Mercaptopyruvate *- Pyruvate + H2S 



SO that part of the inhibition might be attributed to removal of this inter- 

 mediate by the arsenite, which is supported by the fact that NH3 produc- 

 tion is inhibited much less than the formation of HgS. In lipoate-dependent 

 enzyme systems it is often difficult to determine if the site of arsenical 

 inhibition is the apoenzyme or the lipoate (see page 651). The pentavalent 

 arsenicals may oxidize SH groups and occasionally inhibition is reversed 

 by reducing agents. Thus Itoh et al. (1939) found that serum lipase in- 

 hibited by atoxyl can be partially reactivated by cyanide. 



Arsenite may affect enzyme activity by a nonspecific anionic action, 

 as in the stimulation of salt-free fumarase (Massey, 1953 a), or by a more 

 specific competitive inhibition with an anionic substrate, as in the depression 

 of nitrite oxidation in Nitrobacter (Butt and Lees, 1960). Arsenite might 

 act as a reductant in certain oxidizing systems and thus inhibit. The for- 

 mation of (3-chlorolevulinate from /5-ketoadipate and Ch by a soluble 

 chlorinase from Caldariomyces possibly involves a chlorinium ion, arising 

 by oxidation of CI" by oxygen, and the inhibition by arsenite was suggested 

 as resulting from its reducing power rather than a reaction with SH groups 

 (Shaw and Hager, 1959). The organic arsenicals can inhibit by a mechanism 

 more related to their total structure, either competing with a substrate 

 of similar properties or simply binding at or near the active site through 

 a variety of forces. Benzoates and other aromatic substances often inhibit 

 quite effectively and there is no reason that aromatic arsenicals cannot act 

 in the same manner. Some likely instances of this type of inhibition will 

 be mentioned in connection with specific enzymes (see page 647). If inhi- 

 bition of an enzyme is observed at arsenical concentrations only around 

 1 mM or higher, at least one must seriously consider these other mechanisms, 

 but even potent effects do not establish an SH mechanism. 



Use of Arsenicals to Detect Monothiol and Dithiol Groups on Enzymes 



The arsenoso and arsinoso compounds have come to be tools for the de- 

 tection of single SH groups and vicinal pairs of SH groups on enzymes, in- 

 asmuch as arsenoso (or arsenoxide) derivatives are supposed to react with 

 two close SH groups preferentially while the arsinoso (or arsinous acid) 

 derivatives are supposed to react only with single SH groups. The basis 

 for this is principally the work on the reactions of arsenicals with simple 

 monothiols and dithiols, as discussed previously (page 612). Various criteria 

 are used for concluding that an enzyme has single or paired SH groups. 

 The initial distinction was made by Lotspeich and Peters (1951) in their 

 study of isocitrate dehydrogenase, which responds to arsenicals quite dif- 

 ferently than the «-keto acid dehydrogenases. The latter are more sensitive 



