title: Pyruvate Occupancy in the Carboxyl Transferase Domain of Pyruvate Carboxylase Facilitates Product Release from the Biotin Carboxylase Domain through an Intermolecular Mechanism.
abstract: Protein structure, ligand binding, and catalytic turnover contributes to the governance of catalytic events occurring at spatially distinct domains in multifunctional enzymes. Coordination of these catalytic events partially rests on the ability of spatially discrete active sites to communicate with other allosteric and active sites on the same polypeptide chain (intramolecular) or on different polypeptide chains (intermolecular) within the holoenzyme. Often, communication results in long-range effects on substrate binding or product release. For example, pyruvate binding to the carboxyl transferase (CT) domain of pyruvate carboxylase (PC) increases the rate of product release in the biotin carboxylase (BC) domain. In order to address how CT domain ligand occupancy is "sensed" by other domains, we generated functional, mixed hybrid tetramers using the E218A (inactive BC domain) and T882S (low pyruvate binding, low activity) mutant forms of PC. The apparent Ka pyruvate for the pyruvate-stimulated release of Pi catalyzed by the T882S:E218A[1:1] hybrid tetramer was comparable to the wild-type enzyme and nearly 10-fold lower than that for the T882S homotetramer. In addition, the ratio of the rates of oxaloacetate formation to Pi release for the WT:T882S[1:1] and E218A:T882S[1:1] hybrid tetramer-catalyzed reactions was 0.5 and 0.6, respectively, while the T882S homotetramer exhibited a near 1:1 coupling of the two domains, suggesting that the mechanisms coordinating catalytic events is more complicated that we initially assumed. The results presented here are consistent with an intermolecular communication mechanism, where pyruvate binding to the CT domain is "sensed" by domains on a different polypeptide chain within the tetramer.
authors.affiliation: Department of Biochemistry and Molecular Biology and the ‡East Carolina Diabetes and Obesity Institute, Brody School of Medicine at East Carolina University , Greenville, North Carolina 27834, United States.
authors.forename: Lauren E
authors.initials: LE
authors.lastname: Westerhold
authors.affiliation: Department of Biochemistry and Molecular Biology and the ‡East Carolina Diabetes and Obesity Institute, Brody School of Medicine at East Carolina University , Greenville, North Carolina 27834, United States.
authors.forename: Stephanie L
authors.initials: SL
authors.lastname: Adams
authors.affiliation: Department of Biochemistry and Molecular Biology and the ‡East Carolina Diabetes and Obesity Institute, Brody School of Medicine at East Carolina University , Greenville, North Carolina 27834, United States.
authors.forename: Hanna L
authors.initials: HL
authors.lastname: Bergman
authors.affiliation: Department of Biochemistry and Molecular Biology and the ‡East Carolina Diabetes and Obesity Institute, Brody School of Medicine at East Carolina University , Greenville, North Carolina 27834, United States.
authors.forename: Tonya N
authors.initials: TN
authors.lastname: Zeczycki
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism; D001665:Binding Sites; D001710:Biotin / Q000378:metabolism*; D019731:Carbon-Nitrogen Ligases / Q000737:chemistry* / Q000378:metabolism; D019878:Carboxyl and Carbamoyl Transferases / Q000737:chemistry* / Q000378:metabolism; D020134:Catalytic Domain; D018360:Crystallography, X-Ray; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009154:Mutation / Q000235:genetics; D011487:Protein Conformation; D011766:Pyruvate Carboxylase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D019289:Pyruvic Acid / Q000737:chemistry* / Q000378:metabolism; D017386:Sequence Homology, Amino Acid
chemical_list: D001426:Bacterial Proteins; D001710:Biotin; D019289:Pyruvic Acid; D019878:Carboxyl and Carbamoyl Transferases; D019731:Carbon-Nitrogen Ligases; C019791:biotin carboxylase; D011766:Pyruvate Carboxylase