| Abstract |
A citrate synthase was purificed from the cell lysate of Aspergillus nidulans FGSC4 by a consecutive purification procedure including chromatography on DEAE-Sephadex A-50, on Reactive Blue 4 agarose, and on Superose 12 HR column, and then the physical and enzymatic characteristics of the citrate synthase were analyzed. The molecular weight of the enzyme subunit was shown to be 48.2 kDa by SDS-polyacrylamide gel electrophoresis. The optimal pH value for the enzyme activity was pH 8.0, and the optimal temperature 55℃ under the assay conditions. While the citrate synthase retained its activity at 30℃ for at least 1 hr, it lost about 30% and 50% of its activity at 40℃ and 50℃ in 1 hr, respectively. At 60℃, the enzyme lost 80% of its activity in 15 min. The enzyme showed high stability in the range of pH 6.0~7.5 at least for 1 hr. At the concentration of 1 mM, Zn^2- ion inhibited 90% of the enzyme activity, and Mn^2+, Co^2+, and Cu^2+ ions by 20~60%. On the contrary, slightly increased activity was detected in the presence of 1 mM of Mg^2+ ion. Among the seveal plausible regulatory metabolites tested, ATP showed the trongest inhibitory effect on the enzyme. At the concentration of 10mM, ATP inhibit 60% of the enzyme activity; ADP, NADH and NADPH 25~30%; and AMP, NAD^+, NADP^+, fructose 1,6-bisphosphate, fructose 2,6-bisphosphate and glucose 6-phosphate less than 10%. The K_m value for acetyl-CoA was determined to be 16.5 μM, and that for oxaloacetate to be 40.5μM. |
