Regulation of Citrate Synthase in Brevibacterium flavum, a Glutamate-Producing Bacterium
1977 Volume 82 Issue 2 Pages 395-405
Details
1977 Volume 82 Issue 2 Pages 395-405
Citrate synthase [EC 4. 1. 3. 7] was purified about 50-fold from the sonicate of Brevibacterium flavum, a glutamate-producing bacterium. The purified enzyme was irreversibly inactivated in low ionic strength buffer but was stabilized by the addition of inorganic salts or of oxalacetate, a substrate of the enzyme. The enzyme had a molecular weight of 92, 000 and an optimum pH of 8.0. The enzyme was activated about 10-fold by 50mM monovalent cations (Li+, Na+, K+, NH4+, Tris+) as well as by 5mM divalent cations (Mg2+, Cat2+).
Kinetic analysis indicated that the enzymatic reaction proceeded by a rapid equilibrium random Bi Bi mechanism with two independent substrate-binding sites for oxalacetate and acetyl-CoA. The Michaelis constants for oxalacetate and acetyl-CoA were 3.9μM and 54μM, respectively, and the dissociation constants for the reaction products, citrate and CoA, were 4.5mM and 0.21mM, respectively.
The enzyme was specifically inhibited by ATP or by cis-aconitate and isocitrate but not by 2-oxoglutarate or NADH. No synergistic interaction between ATP and cis-aconitate was oberved. The extent of inhibition by 5mM cis-aconitate was about 90 per cent at pH 7.0 and 50 per cent at pH 8.0; it was partially competitive with respect to oxalacetate and non-competitive with respect to acetyl-CoA. The inhibitor constant for cis-aconitate was 0.31mM at pH 7.0 and 0.79mM at pH 8.0. Similar cis-aconitate inhibition was observed with citrate synthases of Escherichia coli, Bacillus subtilis, and pig heart.
ATP inhibited the enzyme about 50 per cent at a concentration of 5mM and showed cooperativity at lower concentrations. The inhibition was partially competitive with respect to acetyl-CoA and non-competitive with respect to oxalacetate. The inhibitor constant for ATP was 1.9mM. Based on these results, a sequential feedback control mechanism including cis-aconitate inhibition of citrate synthase was proposed for glutamate biosynthesis in B. flavum.
