Assay and purification of the adenosylcobalamin-dependent 2-methyleneglutarate mutase from Clostridium barkeri

A continuous spectrophotometric assay was developed for the adenosylcobalamin-dependent 2-methyleneglutarate mutase from Clostridium barkeri. Thereby the product (R)-3-methylitaconate is converted by the delta-isomerase from the same organism to 2,3-dimethylmaleate which absorbs at 240 nm, much higher than both parent compounds (delta epsilon = 3.7 mM-1.cm-1). In addition a discontinuous assay using the facile formation of 2,3-dimethylmaleic anhydride in aqueous solution at pH 0-1 (delta epsilon = 4.0 mM-1.cm-1 at 256 nm) was established. The mutase and the isomerase were purified together by chromatography on quaternary-amine-Sepharose (Q-Sepharose) and on cyanocobalamin-agarose. The enzymes were separated and obtained in homogenous forms by preparative PAGE in non-denaturing buffer. Both enzymes appear to be homotetramers with subunits of 70 kDa (mutase) and 50 kDa (isomerase). The equilibrium constants for both reactions were determined at I = 0.1 M and 25 degrees C: K1, app = [(R)-3-methylitaconate].[2-methyleneglutarate]-1 = 0.26 +/- 0.04, K2,app = [2,3-dimethylmaleate].[(R)-3-methylitaconate]-1 = 7.40 +/- 0.21.