title: Active site mutants of Escherichia coli citrate synthase. Effects of mutations on catalytic and allosteric properties.
abstract: We report properties of five active site mutants of Escherichia coli citrate synthase, in which histidine 264, aspartate 362, and phenylalanine 383 were replaced by alanines, and arginines 387 and 407 by leucines. All mutants have much lower turnover numbers than wild type enzyme; the strongest effect was with the arginine 387 mutant, perhaps because the substrate, oxaloacetate, binds in a different orientation. The arginine 407 mutant has lost most of its ability to distinguish alpha-ketoglutarate, a competitive inhibitor, from oxaloacetate. The mutations of histidine 264 and aspartate 362 affect steady-state kinetics as would be anticipated from current models for citrate synthase catalysis, and resemble mutations of these residues, in pig heart and E. coli enzyme, reported by others. Mutations of residues 264, 362, and 383 also affect allosteric properties. With the phenylalanine 383 mutant, acetyl-CoA saturation is strongly sigmoid, even in the presence of the activator, KCl, implying a marked shift of the allosteric equilibrium toward the T state. The histidine 264 mutant appears to be shifted toward R state and shows weaker binding of the allosteric inhibitor, NADH; thus this mutation also affects the allosteric site, 25-30 A away.
authors.affiliation: Department of Chemistry, University of Manitoba, Winnipeg, Canada.
authors.forename: D S
authors.initials: DS
authors.lastname: Pereira
authors.forename: L J
authors.initials: LJ
authors.lastname: Donald
authors.forename: D J
authors.initials: DJ
authors.lastname: Hosfield
authors.forename: H W
authors.initials: HW
authors.lastname: Duckworth
mesh_terms: D000105:Acetyl Coenzyme A / Q000378:metabolism; D000494:Allosteric Regulation; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D002384:Catalysis; D002950:Citrate (si)-Synthase / Q000235:genetics* / Q000378:metabolism; D004269:DNA, Bacterial; D004926:Escherichia coli / Q000201:enzymology*; D008969:Molecular Sequence Data; D009154:Mutation
chemical_list: D004269:DNA, Bacterial; D000105:Acetyl Coenzyme A; D002950:Citrate (si)-Synthase