Mechanism of Pigeon Liver Malic Enzyme. Modification of Sulfhydryl Groups by 5,5′-Dithiobis(2-nitrobenzoic Acid) and N-Ethylmaleimide

Abstract

Abstract Malic enzyme of pigeon liver (l-malate:NADP+ oxidoreductase (decarboxylating) EC 1.1.1.40) contains a total of approximately 10 —SH groups per subunit, reactive with 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB). Two or more exposed groups (Class I) are highly reactive with DTNB; alteration of these groups does not impair the oxidative decarboxylase activity. This activity, however, is lost upon reaction of a less accessible —SH group (Class II), the alteration of which also results in concomitant increase in the ability of this enzyme to reduce pyruvate in the presence of NADPH. The remaining —SH groups (Class III) in the undenatured enzyme are largely unreactive with this reagent. The alkylating reagent N-ethylmaleimide (NEM) induced similar changes in the over-all and reductase activities of this enzyme. These effects by NEM are also attributed to the alteration of Class II —SH group, since they are prevented by reversible attachment of a thionitrobenzoate substituent (from DTNB) to this —SH group prior to reaction with NEM. Modification of Class II —SH group by either reagent is promoted by carboxylic acid substrates, and partially prevented by Mn2+. This group appears to be located at or near the active center of this enzyme, but is not directly involved in the binding of reactants nor in the catalytic process. An —SCN derivative was prepared by reacting the DTNB-modified enzyme with KCN. This derivative was found to be completely active in oxidative decarboxylation. Apparently, the geometry of the active center is such that this activity is impaired by the attachment of a relatively bulky or charged substituent to the Class II —SH group. Furthermore, our experimental data implicate that modification results in the blockage of the reversible C—C bond cleavage step, thereby accounting for the loss of oxidative decarboxylase and decarboxylase activities.