INHIBITION OF ENZYMES 343 



groups involved in affecting oxygenation, and as a consequence the succin- 

 imide ring is opened to form an iV-ethylsuccinamate derivative (Benesch 

 and Benesch, 1961): 



Hb-S Hb— S 



HC— CO HC— CO HC— coo" 



Hb-SH + II N-Et »- I V-Et »- I 



HC— CO HoC-CO H,C-CONH— Et 



Since iV-ethylmaleimide is catalytically hydrolyzed in the presence of 

 imidazole in neutral solution, it was postulated that a favorably situated 

 imidazole ring adjacent to the reactive SH group is responsible for the 

 second reaction. Guidotti and Konigsberg (1964) report that iV-ethyl- 

 maleimide reacts rather readily with the terminal amino group of the 

 a-chain but not with the amino group of the /?-chain when incubation is 

 carried out at pH 7.15, 25°, and 1.8 mM reagent for 1 hr. Thus certain 

 amino groups can react with iV-ethylmaleimide under conditions often 

 used in enzyme work, and specificity toward SH groups cannot be assumed 

 in all cases. 



INHIBITION OF ENZYMES 



It would be expected on the basis of what is known of the reactions of 

 iV-ethylmaleimide with proteins that the rate of enzyme inhibition would 

 seldom be rapid. The inactivation of xylulose-5-P epimerase proceeds 

 steadily over 40 min and would probably continue further (Tabachnick 

 et al., 1958), and alcohol dehydrogenase is inhibited even more slowly 

 (Witter, 1960). Phosphoglucomutase was found to be inhibited by iV- 

 ethylmaleimide more slowly than by any of the other SH reagents, around 

 50% inhibition being seen in 25 min and complete inhibition at 2 hr (Mil- 

 stein, 1961). It requires 90 min to achieve full inactivation of peniciUinase 

 by iV-ethylmaleimide, but p-MB takes only 10 min (J. T. Smith, 1963 a). 

 In some instances a certain degree of inhibition is reached rapidly and 

 further inhibition occurs slowly, as with lipase, 21% inhibition being ob- 

 served after 15 min and only 47% after 18 hr (Ory et al, 1960). The rate 

 of inhibition depends strongly on pH, as expected. Adenylate kinase re- 

 quires 30 min to be maximally inactivated by 0.2 mM A^-ethylmaleimide 

 at pH 7.5, but at pH 9 a concentration of 0.05 mM gives the same inacti- 

 vation in 5 min (Gregory, 1955). The situation with penicillinase is con- 

 fusing, since, J. T. Smith (1963 a) stated that A-ethylmaleimide is more 

 effective at pH 7.4 than at pH 6, but in another report (J. T. Smith, 1963 b) 

 gave data indicating that it was about 10 times more inhibitory at pH 6 

 than at pH 7.4. If the inhibition is due to reaction with enzyme SH groups, 

 one would anticipate an increased effectiveness with an increase in the pH. 



