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The prokaryotic ATP-citrate lyase is considered to be a key enzyme of the carbon dioxide-fixing reductive tricarboxylic acid (RTCA) cycle. Kinetic examination of the ATP-citrate lyase from the green sulfur bacterium Chlorobium limicola (Cl-ACL), an a4b4 heteromeric enzyme, revealed that the enzyme displayed typical Michaelis-Menten kinetics toward ATP with an apparent Km value of 0.21 ± 0.04 mM. However, strong negative cooperativity was observed with respect to citrate binding, with a Hill coefficient (nH) of 0.45. . | Eur. J. Biochem. 269 3409-3416 2002 FEBS 2002 doi 10.1046 j.1432-1033.2002.03016.x Kinetic and biochemical analyses on the reaction mechanism of a bacterial ATP-citrate lyase Tadayoshi Kanao Toshiaki Fukui Haruyuki Atomi and Tadayuki Imanaka Department of Synthetic Chemistry and Biological Chemistry Graduate School of Engineering Kyoto University Japan The prokaryotic ATP-citrate lyase is considered to be a key enzyme of the carbon dioxide-fixing reductive tricarboxylic acid RTCA cycle. Kineiic exami nail on 4 the ATP-ci irate lyase from the green sulfur bacterium Chlorobium limicola Cl-ACL an a4p4 heteromeric enzyme revealed that the enzyme displayed typical Michaelis-Menten kinetics toward ATP with an apparent Km value of 0.21 0.04 mM. However strong negative cooperativity was observed with respect to citrate binding with a Hill coefficient nH of 0.45. Although the dissociation constant of the first citrate molecule was 0.057 0.008 mM binding of the first citrate molecule to the enzyme drastically decreased the affinity of the enzyme for the second molecule by a factor of 23. ADP was a competitive inhibitor of ATP with a Ki value of 0.037 0.006 mM. Togetherwith previous findings thatthe enzyme catalyzed the reaction only in the direction of citrate cleavage these kinetic features indicated that Cl-ACL can regulate both the direction and carbon flux of the RTCA cycle in C. limicola. Furthermore in order to gain insight on the reaction mechanism we performed biochemical analyses of Cl-ACL. His273 of the a subunit was indicated to be the phosphorylated residue in the catalytic center as both catalytic activity and phosphorylation of the enzyme by ATP were abolished in an H273A mutant enzyme. We found that phosphorylation of the subunit was reversible. Nucleotide preference for activity was in good accordance with the preference for phosphorylation of the enzyme. Although residues interacting with nucleotides in the succinyl-CoA synthetase from Escherichia coli were
