Purification and properties of D-galactonate dehydratase from Mycobacterium butyricum.
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 Km 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 muM. Zinc-inhibited enzyme can be reactivated by chelating agents. The dehydratase is heat-resistant but dithiothreitol renders it more sensitive on heating.
D004355:Drug Stability; D005690:Galactose / Q000031:analogs & derivatives / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism; D006358:Hot Temperature; D006836:Hydro-Lyases / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D008274:Magnesium / Q000494:pharmacology; D015636:Magnesium Chloride; D008970:Molecular Weight; D009161:Mycobacterium / Q000201:enzymology*; D013379:Substrate Specificity; D013400:Sugar Acids / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism
D013400:Sugar Acids; D015636:Magnesium Chloride; C012991:galactonic acid; D006836:Hydro-Lyases; C019607:galactonate dehydratase; D008274:Magnesium; D005690:Galactose