Publications

• Catalytic strategy of citrate synthase: subunit interactions revealed as a consequence of a single amino acid change in the oxaloacetate binding site.

  Kurz L.C., Shah S., Frieden C., Nakra T., Stein R.E., Drysdale G.R., Evans C.T., Srere P.A.

  The active site of pig heart citrate synthase contains a histidine residue (H320) which interacts with the carbonyl oxygen of oxaloacetate and is implicated in substrate activation through carbonyl bond polarization, a major catalytic strategy of the enzyme. We report here the effects on the catal ... >> More

  The active site of pig heart citrate synthase contains a histidine residue (H320) which interacts with the carbonyl oxygen of oxaloacetate and is implicated in substrate activation through carbonyl bond polarization, a major catalytic strategy of the enzyme. We report here the effects on the catalytic mechanism of changing this important residue to glycine. H320G shows modest impairment in substrate Michaelis constants [(7-16)-fold] and a large decrease in catalysis (600-fold). For the native enzyme, the chemical intermediate, citryl-CoA, is both hydrolyzed and converted back to reactants, oxaloacetate and acetyl-CoA. In the mutant, citryl-CoA is only hydrolyzed, indicating a major defect in the condensation reaction. As monitored by the carbonyl carbon's chemical shift, the extent of oxaloacetate carbonyl polarization is decreased in all binary and ternary complexes. As indicated by the lack of rapid H320G--oxaloacetate catalysis of the exchange of the methyl protons of acetyl-CoA or the pro-S-methylene proton of propionyl-CoA, the activation of acetyl-CoA is also faulty. Reflecting this defect in acetyl-CoA activation, the carboxyl chemical shift of H320G-bound carboxymethyl-CoA (a transition-state analog of the neutral enol intermediate) fails to decrease on formation of the H3020G-oxaloacetate-carboxymethyl-CoA ternary complex. Progress curves and steady-state data with H320G using citryl-CoA as substrate show unusual properties: substrate inhibition and accelerating progress curves. Either one of two models with subunit cooperativity [Monod, J., Wyman, J., & Changeux, J.-P. (1965) J. Mol. Biol. 12, 88; Koshland, D. E., Jr., Nemethy, G., & Filmer, D. (1966) Biochemistry 5, 365] quantitatively accounts for both the initial velocity data and the individual progress curves. The concentrations of all enzyme forms and complexes are assumed to rapidly reach their equilibrium values compared to the rate of substrate turnover. The native enzyme also behaves according to models for subunit cooperativity with citryl-CoA as substrate. However, the rates of formation/dissociation and reaction of complexes are kinetically significant. Comparisons of the values of kinetic constants between the native and mutants enzymes lead us to conclude that the mutant less readily undergoes a conformation change required for efficient activation of substrates. << Less

  Biochemistry 34:13278-13288(1995) [PubMed] [EuropePMC]

• Proposed mechanism for the condensation reaction of citrate synthase: 1.9-A structure of the ternary complex with oxaloacetate and carboxymethyl coenzyme A.

  Karpusas M., Branchaud B., Remington S.J.

  The crystal structure of the ternary complex citrate synthase-oxaloacetate-carboxymethyl coenzyme A has been solved to a resolution of 1.9 A and refined to a conventional crystallographic R factor of 0.185. The structure resembles a proposed transition state of the condensation reaction and sugges ... >> More

  The crystal structure of the ternary complex citrate synthase-oxaloacetate-carboxymethyl coenzyme A has been solved to a resolution of 1.9 A and refined to a conventional crystallographic R factor of 0.185. The structure resembles a proposed transition state of the condensation reaction and suggests that the condensation reaction proceeds through a neutral enol rather than an enolate intermediate. A mechanism for the condensation reaction is proposed which involves the participation of three key catalytic groups (Asp 375, His 274, and His 320) in two distinct steps. The proposed mechanism invokes concerted general acid-base catalysis twice to explain both the energetics of the reaction and the experimentally observed inversion of stereochemistry at the attacking carbon atom. << Less

  Biochemistry 29:2213-2219(1990) [PubMed] [EuropePMC]

• Active site mutants of pig citrate synthase: effects of mutations on the enzyme catalytic and structural properties.

  Evans C.T., Kurz L.C., Remington S.J., Srere P.A.

  We examined the catalytic efficiency of 18 pig citrate synthase mutants. The residues mutated were selected according to two criteria: the conservation of that residue in all known citrate synthase sequences, and the importance of that residue in substrate-amino acid interactions suggested by the ... >> More

  We examined the catalytic efficiency of 18 pig citrate synthase mutants. The residues mutated were selected according to two criteria: the conservation of that residue in all known citrate synthase sequences, and the importance of that residue in substrate-amino acid interactions suggested by the extensive crystal structure information on the enzyme and its complexes. Several changes were made at certain residues to probe the effects of size, hydrogen bonding, and charge on the kinetics of the enzyme. The mutations, as expected, affected the kcats and Kms for OAA and acetyl-CoA to varying degrees. The catalytic efficiency of each of the mutants was determined by the kcat/Km for the individual substrates, OAA and acetyl-CoA. All mutations affected kcat. There was only one mutant, Asp327 Asn, in which the Kms primarily were affected. Most mutations affected both kcat and Km and included the following: His274Gly, His274Arg, Asp375Gly, Asp375Asn, Asp375Glu, Asp375Gln, His320Gly, His320Gln, His320Asn, His320Arg, Arg401His, Gly275Val, and Gly275Ala. The mutations, Arg401Gly, Arg401Lys, His235Gln, and Asn242Glu, had smaller effects on kcat and Km. The CS mutant Arg401Lys exhibited a modestly improved kcat/Km for both substrates compared to the nonmutant enzyme. X-ray crystallographic studies at 2.7 A resolution of one of the mutants, His274Gly, have been undertaken. The mutant enzyme crystallizes in an "open" conformation essentially isomorphous to wild type. The refined model has good geometry and a crystallographic R factor of 0.187 for 11 441 reflections observed between 6.0 and 2.7 A resolution. The refined model revealed a localized relaxation of the structure to relieve strain imposed by a high-energy main and side chain conformation of His274 in the nonmutant, but otherwise the mutation does not result in major structural alterations. Preliminary electrostatic calculations provide support for the concept that the transition state in the rate-limiting step of the citrate synthase catalyzed reaction may be an "enolized" version of acetyl-CoA that is neither neutral nor fully negatively charged and that a possible role for the catalytically essential His274 is to stabilize this by charge delocalization mediated by a hydrogen bond. These results provide the basis for further studies of the effects of these changes on the several reactive intermediates, activated substrates, and transition states which may occur along the reaction coordinate for this type of Claisen enzyme. << Less

  Biochemistry 35:10661-10672(1996) [PubMed] [EuropePMC]

• Purification and characterization of citrate synthase from Methylobacterium extorquens--a methylotrophic producer of polyhydroxybutyrate.

  Belova L.L., Sokolov A.P., Morgunov I.G., Trotsenko YuA

  Citrate synthase (citrate oxaloacetate-lyase, CoA-acetylating; EC 4.1.3.7, CS) was isolated and purified to homogeneity from a methylotrophic producer of polyhydroxybutyrate (PHB), Methylobacterium extorquens 15. The purification procedure includes streptomycin sulfate treatment of cell-free extra ... >> More

  Citrate synthase (citrate oxaloacetate-lyase, CoA-acetylating; EC 4.1.3.7, CS) was isolated and purified to homogeneity from a methylotrophic producer of polyhydroxybutyrate (PHB), Methylobacterium extorquens 15. The purification procedure includes streptomycin sulfate treatment of cell-free extract, ammonium sulfate fractionation, two steps of hydrophobic chromatography, and ion-exchange chromatography. The specific activity of the final enzyme preparation was 24 U/mg protein. The enzyme has apparent molecular weight 260 kD and consists of four 66-kD subunits. The enzyme shows a sigmoid saturation curve with CoASA (h = 1.3). Kinetic parameters are: K(m) = 84 microM for CoASA; K(m) = 12 microM for oxaloacetate; Vmax = 29.7 mumoles/min per mg protein. KCl at concentrations up to 80 mM activates the CS. ATP exerts a significant inhibitory effect on the enzyme activity, whereas NAD(P)H, isocitrate, alpha-ketoglutarate, ADP, acetoacetyl-CoA, glyoxylate, and glutamate have no influence. A possible role of the CS in coordinated control of CoASA transformation through the tricarboxylic acid cycle and PHB biosynthesis in this methylotroph is discussed. << Less

  Biochemistry (Mosc) 62:71-76(1997) [PubMed] [EuropePMC]