INHIBITION OF ENZYMES 717 



to the ionization of the SH groups. Penicillinase is inhibited 29% by 2 xnM 

 o-iodosobenzoate at pH 7.4 but not at all at pH 6 (Smith, 1963 b). On the 

 basis of these results, one might conclude that one should avoid pH's of 

 7 or below in enzyme work. Unfortunately, there are no data on the speci- 

 ficity of o-iodosobenzoate at higher pH's. 



Protection of Enzymes against Inhibition by o-lodosobenzoate 



Protection of an enzyme by addition of some thiol with the o-iodoso- 

 benzoate does not tell one anything about the mechanism of inhibition, 

 since the inhibitor is simply depleted by oxidation of the thiol. However, 

 protection by substances interacting with the enzyme active center pro- 

 vides some evidence for the site of the inhibition by o-iodosobenzoate. The 

 normal substrate of an enzyme has been shown frequently to protect against 

 o-iodosobenzoate, if it is present during the incubation of the enzyme with 

 the inhibitor. Thus, alcohol dehydrogenase is protected by ethanol (Barron 

 and Levine, 1952), fumarase by either fumarate or malate (Favelukes and 

 Stoppani, 1958), D-amino acid oxidase by alanine (Frisell and Hellerman, 

 1957), choline oxidase by choline (Rothschild et al., 1954), glutamate se- 

 mialdehyde reductase by glutamate semialdehyde (Smith and Greenberg, 

 1957), succinate oxidase by succinate (Thorn, 1959), and homogentisicase 

 by homogentisate (Tokuyama, 1959). In some instances the protection may 

 be very marked; e.g., fumarase is completely protected against 0.5 vaM 

 o-iodosobenzoate by 25 mM fumarate, and a 58% inhibition of alcohol dehy- 

 drogenase is reduced to 6.5% by ethanol. Coenzymes can likewise protect 

 in certain instances: aldehyde dehydrogenase is protected by NAD and 

 NADP (Stoppani and Milstein, 1957 a,b), alcohol dehydrogenase is protect- 

 ed by NAD (Barron and Levine, 1952), and D-amino acid oxidase is pro- 

 tected by FAD (Frisell and Hellerman, 1957). It is interesting that the dif- 

 ferent aldehyde dehydrogenases are protected to different degrees by their 

 coenzymes. The K+-activated yeast enzyme is protected by both NAD and 

 NADP, as well as by acetaldehyde, whereas the NAD-linked liver enzyme 

 is protected only by NAD and not by NADP. These observations may be 

 taken to mean that the inhibition is the result of a reaction of o-iodoso- 

 benzoate with SH groups at or near the active center, and that when the 

 active center is covered by substrate or coenzyme the oxidant is unable to 

 attack these SH groups. Such effects must be taken into account when 

 o-iodosobenzoate is used in cellular preparations. In one instance the effect 

 of substrate is abnormal. The lactate dehydrogenase of Propionibacterium 

 pentosaceum is inhibited more readily in the presence of lactate than in its 

 absence (see accompanying tabulation) (Molinari and Lara, 1960). This 

 was explained by assuming that lactate increases the fraction of free SH 

 groups, suggesting that the SH groups may be involved in the electron 

 transport. 



