344 3. .Y-ETHYLMALEIMIDE 



In any event, enzyme inhibition with lY-ethylmaleimide is generally slow 

 and more attention should be paid to the kinetics in studies using this 

 substance to detect SH groups on enzymes, and it is likely that some of 

 the failures to observe inhibition are due to insufficient incubation time. 

 Thus the data given in Table 3-1 must be used with some caution. In 

 most cases the inhibition by A'^-ethylmaleimide is probably irreversible, 

 although this has not been commonly investigated. The inhibition of leu- 

 cine aminopejitidase is not reversed by cysteine (Green et al., 1955), and 

 the inhibition of glutathione reductase is not reversed by dialysis (Mapson 

 and Isherwood, 1963). 



iV-Ethylmaleimide sometimes reacts with SH groups which are at or 

 near the binding sites for coenzymes or other components of the enzyme 

 reaction. Incubation of the L( + )-lactate dehydrogenase of yeast with 

 iV-ethylmaleimide leads to some dissociation of flavin from the enzyme 

 (Armstrong et al., 1960), and the binding of NADH to cytochrome 65 

 aporeductase is almost completely prevented by 0.02 mM A^-ethylmaleimide 

 (Strittmatter, 1961 b), although the binding of FMN is not affected. Other 

 evidence comes from protection experiments. NAD and NADH partially 

 protect the K+-activated aldehyde dehydrogenase of yeast against N- 

 ethylmaleimide, while NADP protects the NADP-linked aldehyde dehy- 

 drogenase (Stoppani and Milstein, 1957 b). NADH also protects the liver 

 alcohol dehydrogenase quite effectively (Witter, 1960). The fatty acid- 

 activating enzyme is protected partially by ATP (Jencks and Lipmann, 

 1957), and succinate dehydrogenase is protected to some extent by phos- 

 phate (Stoppani and Brignone, 1956). One cannot certainly conclude that 

 the protector is bound to the SH groups which are reacted by A'-ethyl- 

 maleimide, but it is likely that the SH groups are at least close to the 

 binding sites. The inhibition of glutaminase by A^-ethylmaleimide was 

 stated by Sayre and Roberts (1958) to be more nearly competitive with 

 glutamine than noncompetitive, and the Ijv — [1/(S)] plot, despite some 

 nonlinearity of the curve for the inhibited enzyme, certainly indicates a 

 mixed inhibition, which is probably the result of the nonequilibrium con- 

 ditions (i. e., the glutamine probably is exerting a protective action and 

 delaying the development of the inhibition, the reactive SH group being 

 vicinal to the glutamine site). 



Glutathione reductase presents an interesting situation since incuba- 

 tion of the enzyme with A-ethylmaleimide in the absence of NADH or 

 NADPH does not result in inhibition, whereas the presence of NADPH 

 allows the A^-ethylmaleimide to react with the enzyme (Black and Hudson, 

 1961). It was suggested that NADPH must expose one or more SH groups 

 when it is bound to the enzyme, and it was demonstrated with A^-ethyl- 

 maleimide-C^* that more of the inhibitor is attached to the enzyme when 

 NADPH is present. The SH groups exposed are not those of the lipoate 



