Purification and properties of d-galactonate dehydratase from mycobacterium butyricum

Abstract

d-Galactonate dehydratase (d-galactonate hydro-lyase, EC 4.2.1.6) catalyzes the first reaction in the d-galactonate catabolic pathway of non-pathogenic Mycobacteria. As a part of studies concerning the metabolism of d-galactose and related compounds as well as its regulation in saprophytic strains of Mycobacteria, d-galactonate dehydratase has been purified and enzymologically characterized. The enzyme has been purified 325-fold from the crude extracts of galactose-grown Mycobacterium butyricum and its molecular weight of about 270 000 has been determined by Sephadex G-200 filtration. Isolation and analysis procedures are described. The dehydratase reaction is optimal within a pH range of 7.8–8.0. The enzyme is strictly specific for d-galactonate; none of the other sugar acids tested serves as a substrate or inhibits the dehydration of d-galactonate. The K_m value for d-galactonate is 1 mM. The enzyme requires Mg²⁺ or Mn²⁺ for activity. The dehydratase is very sensitive to SH-blockers; the most potent inhibitor is ZnSO₄, which considerably inhibits the enzyme at a concentration of 2.5–5.0 μM. Zinc-inhibited enzyme can be reactivated by chelating agents. The dehydratase is heat-resistant but dithiothreitol renders it more sensitive on heating.