title: Structural basis for regulation in gram-negative bacterial citrate synthases.
abstract: The citrate synthases of Gram-negative bacteria, unlike those of eukaryotes, are inhibited allosterically by NADH, but the two kinds of citrate synthase are about 30% homologous in amino acid sequence--the two Gram-negative citrate synthase sequences so far determined, from Escherichia coli and Acinetobacter anitratum, are about 70% identical. A model for the NADH-sensitive E. coli citrate synthase has been constructed using sequence homology and the known structure of the pig heart enzyme. The most reactive cysteine in the E. coli enzyme, which probably marks the NADH binding site, has now been identified as Cys-206. The model places this residue far from the active site. An E. coli citrate synthase mutant, from which a stretch of 24 amino acids has been deleted near the active site, still binds NADH normally. Two active site missense mutants of this enzyme, generated by oligonucleotide-directed mutagenesis, have lower affinities for one substrate, oxaloacetate, but also are much less sensitive to 2-oxoglutarate, an oxaloacetate analogue hitherto believed to be an allosteric inhibitor. These results confirm that NADH binds to a truly allosteric site in E. coli citrate synthase, the features of which are still to be defined; while 2-oxoglutarate is really an active-site directed inhibitor, although it may still play a regulatory role in vivo.
authors.affiliation: Department of Chemistry, University of Manitoba, Winnipeg, Canada.
authors.forename: H W
authors.initials: HW
authors.lastname: Duckworth
authors.forename: D H
authors.initials: DH
authors.lastname: Anderson
authors.forename: A W
authors.initials: AW
authors.lastname: Bell
authors.forename: L J
authors.initials: LJ
authors.lastname: Donald
authors.forename: A L
authors.initials: AL
authors.lastname: Chu
authors.forename: G D
authors.initials: GD
authors.lastname: Brayer
mesh_terms: D001665:Binding Sites; D002950:Citrate (si)-Synthase / Q000378:metabolism*; D002952:Citric Acid Cycle; D006090:Gram-Negative Bacteria / Q000378:metabolism*; D007656:Ketoglutaric Acids / Q000378:metabolism; D008958:Models, Molecular; D007652:Oxo-Acid-Lyases / Q000378:metabolism*; D011487:Protein Conformation
chemical_list: D007656:Ketoglutaric Acids; D002950:Citrate (si)-Synthase; D007652:Oxo-Acid-Lyases