PHENOL OXIDASES 301 



reich, a marked decrease in the inhibition with an elevation of the pH 

 from 5.3 to 7 is observed, but Krueger interpreted this as indicating an 

 ionizing group on the enzyme with a pK^ around 6 and possibly an imida- 

 zole group. One reason for assuming the ionizing group to be on the enzyme 

 is the pH effect on inhibitions by the inorganic anions; however, the in- 

 hibition by these ions may be through a different mechanism than the 

 benzoates, and indeed Krueger found noncompetitive kinetics for chloride. 

 The substrates for the enzyme are, of course, un-ionized and this might 

 favor the concept of the acid form of the inhibitors being active and the 

 important ionizing group on the inhibitors. It is impossible at the present 

 time to decide which is the correct interpretation and hence the signifi- 

 cance of the pH effects. Ionizing groups on both enzyme and inhibitors 

 might also be considered. It should be added that the following were found 

 to be without effect: sulfate (40 mM), nitrate (40 mM), pyrophosphate (40 

 mM), pyruvate (20 mM), succinate (20 mM), maleate (20 mM), fumarate 

 (20 mM), and ethyl benzoate (4 mM). 



The lack of information on the exact catalytic mechanism involved in 

 these enzymes, and particularly our ignorance of the state and role of 

 copper, make it difficult to understand the binding of the inhibitors. There 

 are two copper ions at the active center but we do not know if they com- 

 plex with oxygen, or the substrates, or both, or whether one copper com- 

 plexes with oxygen and the other with the substrates. Some of the ionic 

 inhibitions might be due to the formation of complexes with the copper; 

 such complexes might be more difficult to form at higher pH's because of 

 competition with hydroxyl ions. The strong inhibition by oxalate might 

 be due to chelation of the copper, but it is odd that pyrophosphate does 

 not inhibit since it also chelates copper weU. It is also surprising that sul- 

 fate and nitrate do not inhibit at all, unless the ionic size is as critical as 

 Krueger believes. 



It would appear that inhibitory activity is related to the presence of 

 a carboxyl group (excepting the inorganic ions). Benzoate ester and benza- 

 mide are bound much less tightly than benzoate (around 3 kcal/mole 

 difference). The weak action of benzenesuLfonate might be explained in 

 three ways; (1) if the un-ionized form of the acid is necessary for activity, 

 there would be less in the case of benzenesulfonic acid than with benzoic 

 acid, (2) the sulfonate group might be too bulky, according to Krueger 

 (although it is certainly not much larger than the carboxyl group), and 

 (3) the sulfonate group does not have the ability to form bonds with the 

 copper or hydrogen bonds with an enzyme group. Separation of the car- 

 boxyl from the benzene ring reduces the binding, as in phenylacetate or 

 cinnamate. The second requirement for potent inhibitory activity is a 

 benzene ring. This may be seen by comparing acetate and phenylacetate, 

 benzoate and nicotinate, and benzoate and cyclohexanecarboxylate; in 



