title: Investigation of the Roles of Allosteric Domain Arginine, Aspartate, and Glutamate Residues of Rhizobium etli Pyruvate Carboxylase in Relation to Its Activation by Acetyl CoA.
abstract: The mechanism of allosteric activation of pyruvate carboxylase by acetyl CoA is not fully understood. Here we have examined the roles of residues near the acetyl CoA binding site in the allosteric activation of Rhizobium etli pyruvate carboxylase using site-directed mutagenesis. Arg429 was found to be especially important for acetyl CoA binding as substitution with serine resulted in a 100-fold increase in the Ka of acetyl CoA activation and a large decrease in the cooperativity of this activation. Asp420 and Arg424, which do not make direct contact with bound acetyl CoA, were nonetheless found to affect acetyl CoA binding when mutated, probably through changed interactions with another acetyl CoA binding residue, Arg427. Thermodynamic activation parameters for the pyruvate carboxylation reaction were determined from modified Arrhenius plots and showed that acetyl CoA acts to decrease the activation free energy of the reaction by both increasing the activation entropy and decreasing the activation enthalpy. Most importantly, mutations of Asp420, Arg424, and Arg429 enhanced the activity of the enzyme in the absence of acetyl CoA. A main focus of this work was the detailed investigation of how this increase in activity occurred in the R424S mutant. This mutation decreased the activation enthalpy of the pyruvate carboxylation reaction by an amount consistent with removal of a single hydrogen bond. It is postulated that Arg424 forms a hydrogen bonding interaction with another residue that stabilizes the asymmetrical conformation of the R. etli pyruvate carboxylase tetramer, constraining its interconversion to the symmetrical conformer that is required for catalysis.
authors.affiliation: Department of Biochemistry, Faculty of Science, Mahidol University , Bangkok 10400, Thailand.
authors.forename: Chaiyos
authors.initials: C
authors.lastname: Sirithanakorn
authors.affiliation: Department of Biochemistry, Faculty of Science, Mahidol University , Bangkok 10400, Thailand.
authors.forename: Sarawut
authors.initials: S
authors.lastname: Jitrapakdee
authors.affiliation: School of Chemistry and Biochemistry, The University of Western Australia , 35 Stirling Highway, Crawley, WA 6009, Australia.
authors.forename: Paul V
authors.initials: PV
authors.lastname: Attwood
mesh_terms: D000105:Acetyl Coenzyme A / Q000378:metabolism; D000494:Allosteric Regulation; D000495:Allosteric Site / Q000235:genetics; D000595:Amino Acid Sequence; D001120:Arginine / Q000737:chemistry; D001224:Aspartic Acid / Q000737:chemistry; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D004789:Enzyme Activation; D018698:Glutamic Acid / Q000737:chemistry; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D011766:Pyruvate Carboxylase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D042163:Rhizobium etli / Q000201:enzymology* / Q000235:genetics
chemical_list: D001426:Bacterial Proteins; D011994:Recombinant Proteins; D001224:Aspartic Acid; D018698:Glutamic Acid; D000105:Acetyl Coenzyme A; D001120:Arginine; D011766:Pyruvate Carboxylase