Nicotinic Acid Metabolism: VI. PURIFICATION AND PROPERTIES OF α-METHYLENEGLUTARATE MUTASE (B12-DEPENDENT) AND METHYLITACONATE ISOMERASE

Abstract

Abstract The conversion of α-methyleneglutarate (αMG) to dimethylmaleate (DMM) via methylitaconate (MeIT) is the reaction sequence that produces a symmetrical intermediate in the fermentation of nicotinic acid by Clostridium barkeri. αMG mutase catalyzes the isomerization of αMG in a B12 coenzyme-dependent reaction to form MeIT which in turn is isomerized to DMM by MeIT isomerase. Both enzymes are induced by growth on nicotinic acid and are not detectable in cells grown on glucose. The isomerase reaction favors the formation of DMM, whereas the mutase reaction favors the formation of αMG. Both reactions were demonstrated to be reversible. αMG mutase activity was assayed by coupling the formation of MeIT with the production of DMM in the presence of the isomerase. The purified mutase has an absolute requirement for cobamide coenzyme. The apparent Km values for αMG, α-(5,6-dimethylbenzimidazolyl)-cobamide coenzyme, α-(benzimidazolyl)-cobamide coenzyme, and α-(adenyl)-cobamide coenzyme are 7.14 x 10-3, 7.3 x 10-8, 3.0 x 10-7, and 1.25 x 10-6m, respectively. The most effective coenzyme, α-(5,6-dimethylbenzimidazolyl)cobamide coenzyme, is the same one that is synthesized in large amounts when C. barkeri is grown on nicotinic acid. No added cofactors are required for methylitaconate isomerase activity. The Km value is 6.2 x 10-3 m for methylitaconate and 4.0 x 10-3 m for dimethylmaleate. Both the mutase and the isomerase appear to be sulfhydryl proteins; neither requires metal ions for activity. A number of dicarboxylic acids are competitive inhibitors of αMG mutase but none of these affect MeIT isomerase activity.