title: Kinetics and mechanism of the citrate synthase from the thermophilic archaeon Thermoplasma acidophilum.
abstract: The kinetics and mechanism of the citrate synthase from a moderate thermophile, Thermoplasma acidophilum (TpCS), are compared with those of the citrate synthase from a mesophile, pig heart (PCS). All discrete steps in the mechanistic sequence of PCS can be identified in TpCS. The catalytic strategies identified in PCS, destabilization of the oxaloacetate substrate carbonyl and stabilization of the reactive species, acetyl-CoA enolate, are present in TpCS. Conformational changes, which allow the enzyme to efficiently catalyze both condensation of acetyl-CoA thioester and subsequently hydrolysis of citryl-CoA thioester within the same active site, occur in both enzymes. However, significant differences exist between the two enzymes. PCS is a characteristically efficient enzyme: no internal step is clearly rate-limiting and the condensation step is readily reversible. TpCS is a less efficient catalyst. Over a broad temperature range, inadequate stabilization of the transition state for citryl-CoA hydrolysis renders this step nearly rate-limiting for the forward reaction of TpCS. Further, excessive stabilization of the citryl-CoA intermediate renders the condensation step nearly irreversible. Values of substrate and solvent deuterium isotope effects are consistent with the kinetic model. Near its temperature optimum (70 degrees C), there is a modest increase in the reversibility of the condensation step for TpCS, but reversibility still falls short of that shown by PCS at 37 degrees C. The root cause of the catalytic inefficiency of TpCS may lie in the lack of protein flexibility imposed by the requirement for thermal stability of the protein itself or its temperature-labile substrate, oxaloacetate.
authors.affiliation: Department of Biochemistry and Molecular Biophysics, Division of Biology and Biomedical Sciences, Washington University School of Medicine, St. Louis, Missouri 63110, USA.
authors.forename: L C
authors.initials: LC
authors.lastname: Kurz
authors.forename: G
authors.initials: G
authors.lastname: Drysdale
authors.forename: M
authors.initials: M
authors.lastname: Riley
authors.forename: M A
authors.initials: MA
authors.lastname: Tomar
authors.forename: J
authors.initials: J
authors.lastname: Chen
authors.forename: R J
authors.initials: RJ
authors.lastname: Russell
authors.forename: M J
authors.initials: MJ
authors.lastname: Danson
mesh_terms: D000105:Acetyl Coenzyme A / Q000737:chemistry; D000214:Acyl Coenzyme A / Q000737:chemistry; D000818:Animals; D002942:Circular Dichroism; D002950:Citrate (si)-Synthase / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D019343:Citric Acid / Q000737:chemistry; D003065:Coenzyme A / Q000737:chemistry; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D007526:Isoelectric Point; D007700:Kinetics; D019906:Nuclear Magnetic Resonance, Biomolecular; D062907:Oxaloacetic Acid / Q000737:chemistry; D011522:Protons; D012997:Solvents; D013379:Substrate Specificity; D013552:Swine; D013696:Temperature; D013822:Thermoplasma / Q000201:enzymology*
chemical_list: D000214:Acyl Coenzyme A; D004791:Enzyme Inhibitors; D011522:Protons; D012997:Solvents; C105313:dethiaacetyl coenzyme A; D019343:Citric Acid; D062907:Oxaloacetic Acid; C029890:citryl-coenzyme A; D000105:Acetyl Coenzyme A; D002950:Citrate (si)-Synthase; D003065:Coenzyme A