INHIBITION OF ENZYMES 465 



of urease even at 18.4 mM, the conclusion being that only the quinone form 

 must be active. Another related approach is to use both the quinone and 

 hydroquinone forms in as pure a state as possible and observe the kinetics 

 of the inhibition. Quastel (1933 b) found, for example, that p-benzoquinone 

 is a more potent inhibitor of urease than p-benzohydroquinone — 0.0037 

 mM of the former inhibited 98% and of the latter 64%. If one compares 

 there results with those of Fischgold, it is clear that Quastel's hydroqui- 

 none sample must have contained quite a bit of quinone; nevertheless, 

 a differential effect like this is indicative if not absolute proof. A somewhat 

 better method is to determine the rates of inhibition, as did Barron (1936) 

 with pyruvate oxidase and Schoetensack (1948) with renal phosphatase, 

 both enzymes being inhibited rapidly by p-benzoquinone but slowly by 

 p-benzohydroquinone, the delayed action in the latter case presumably 

 being due to the necessity for oxidation of the hydroquinone. Since hydro- 

 quinone oxidation rate decreases with a lowering of the pH, one can some- 

 times compare inhibitions more profitably at a reduced pH. Thus Potter 

 (1942) showed that at pH 5 there is a marked differential effect of p-benzo- 

 quinone and p-benzohydroquinone on succinate dehydrogenase — 0.0002 

 mM of the former inhibits 57% after 20 min while the latter does not in- 

 hibit at all. Quastel's experiments were run at pH 7.4 which may account 

 for his results. In most instances the quinone form has been shown to be 

 the more inhibitory, but Franke (1944 b) reported that p-benzoquinone 

 and p-benzohydroquinone inhibit some enzymes equally (oxalate oxidase 

 and lipoxidase), the former to inhibit some more potently (D-amino acid 

 dehydrogenase and succinate dehydrogenase), and the latter to inhibit 

 other enzymes more strongly (catalase and yeast lactate dehydrogenase), 

 although it is not possible to draw definite conclusions since the conditions 

 were not ideal. Some have favored reduction of the enzyme as being re- 

 sponsible for inhibition (Takeuchi, 1933; Itoh et al., 1939), but it is more 

 likely that inhibitions by hydroquinones are related to other mechanisms. 

 The semiquinone form may arise during oxidation-reduction reactions and 

 should at least be considered as the inhibitory species in some cases. It 

 is true that the semiquinones are not as reactive as most free radicals, but 

 they might react with enzymes in ways other than the quinone or hydro- 

 quinone, as Michaelis (1935) emphasized. The participation of the semi- 

 quinone in inhibition will depend not only on the inherent reactivity but 

 as much on the concentration of semiquinone reached in the system, which 

 is dependent on many factors, particularly the pH and the structure of the 

 quinone. This has been studied more carefully in the p-phenylenediamines 

 (Kensler et al, 1942 a) and the inhibition of pyruvate decarboxylase 

 was shown to be correlated with the stability of the semiquinone (see 

 page 590). I know of no direct proof for the importance of the semiquinone 

 in enzyme inhibition. Duroquinone, whose semiquinone is the most stable 



