Abstract
Ammonia-oxidizing chemoautotrophic Nitrosomonas europaea exhibited the NADP-specific isocitrate dehydrogenase (ICDH) activity. Isocitrate-oxidizing activity was found in two fractions (ICDH-FI and ICDH-FII) on a DEAE-Sepharose CL-6B. These enzyme proteins with isocitrate-oxidizing activity were further purified as electrophoretic homogeneity using gel filtration (Cellulofine GCL-2000-m) and then ion exchange chromatography (Mono Q). These enzymes were purified about 69-fold for ICDH-FI and about 140-fold for ICDH-FII. The molecular weights were determined as both 82, 000 for both, using gel filtration and SDS-PAGE of enzymes. The isoelectric points of ICDH-FI and ICDH-FII were pH 5.8 and 6.2, respectively.
The pH and temperature optima for ICDH-FI and ICDH-FII were pH 8.5 and 8.0, respectively, and about 55°C for both enzymes. The enzymes were stable up to 45°C and acted specifically on D- or DL-isocitrate and not on any other organic acids tested. The apparent Km values of ICDH-FI for D- and DL-isocitrate were 15.0μM and 17.5μM, respectively and Km values of ICDH-FII for D- and DL-isocitrate were 13.0μM and 11.5μM, respectively. Both enzymes were strongly activated by Mn2+ and Mg2+, and inhibited by Cu2+, Ni2+, Zn2+ and Hg2+. They were strongly inhibited by p-chloromercuribenzoate.
