Abstract
A cell-free extract of a soil bacterium, Bacillus sp., which catalyzes the oxidation of C5-branched-chain dicarboxylic acids, such as β-methylmalic and citramalic (CMA) acids without adaptation to these acids, was shown to convert (+) CMA into pyruvate and CoASAc through the following two sequential reactions:
(i)(+) CMA+succiny1-CoA←→(+) CMA-CoA+succinate
(ii)(+) CMA-CoA←→pyruvate+CoASAc
These two reactions seemed to be catalyzed by two enzymes and only reaction (ii) was found to require Mg2+ and to be inhibited by EDTA. However, the two enzymes were not separated from one another by ammonium sulfate fractionation, or by DEAE-Sephadex-, or Sephadex G-200 column chromatography.
The cleavage reaction of (+) CMA was inhibited by various anions, and their inhibitory effects decreased in the order of Hoffmeister's series (SCN->NO3->Cl-> SO22-). Dicarboxylic acids, such as succinic acid and its analogues, monocarboxylic acids, and unsaturated dicarboxylic acids of the cis-form strongly inhibited the activity.
The (+) CMA-CoA lyase reaction seems to be reversible because partially purified (+) CMA-cleavage enzymes catalyzed formation of radioactive CMA from 14CCoASAc and pyruvate and because the 14C-CMA formed was dextrorotatory.
The enzymes concerned were demonstrated in cells grown on various organic acids, and so seemed to be constitutive enzymes, unlike those in Pseudomonas.
