Purification And Properties Of D-Gluconate Dehydratase From Clostridium-Pasteurianum

1 d-Gluconate dehydratase from Clostridium pasteurianum has been purified by heat treatment, chromatography on DEAE-Sephadex and gel filtration to about 80% homogeneity. The overall procedure resulted in a 33-fold purification with a yield of 16%. Further purification by electrophoresis yielded preparations homogeneous by electrophoretic and ultracentrifugal criteria. Analysis of the enzyme by sedimentation equilibrium experiments, gel-filtration and sucrosegradient centrifugation gave a molecular weight of 131 000, 130 000 and 118 000, respectively. Treatment with sodium dodecylsulfate and mercaptoethanol dissociated the enzyme into two subunits (Mr= 64000). 2 The enzyme is inactivated by exposure to oxygen and the activity can be restored by incubation with sulfhydryl compounds such as mercaptoethanol, dithiothreitol, reduced glutathione and cysteine in conjunction with ferrous ions for 2 h at 35°C. 3 The reactivated enzyme dehydrates gluconate in the presence of divalent metal ions only. Ferrous ions (2 mM) are most effective but can be partially replaced by Mn2+, Co2+ and Mg2+. EDTA inhibits the reaction completely. 4 d-Gluconate dehydratase exhibits maximum activity in the pH region of 7.3 to 7.8 Among several compounds tested only d-gluconate is dehydrated (Km value = 5.5 mM).