title: Direct Evidence for Metabolon Formation and Substrate Channeling in Recombinant TCA Cycle Enzymes.
abstract: Supramolecular assembly of enzymes into metabolon structures is thought to enable efficient transport of reactants between active sites via substrate channeling. Recombinant versions of porcine citrate synthase (CS), mitochondrial malate dehydrogenase (mMDH), and aconitase (Aco) were found to adopt a homogeneous native-like metabolon structure in vitro. Site-directed mutagenesis performed on highly conserved arginine residues located in the positively charged channel connecting mMDH and CS active sites led to the identification of CS(R65A) which retained high catalytic efficiency. Substrate channeling between the CS mutant and mMDH was severely impaired and the overall channeling probability decreased from 0.99 to 0.023. This work provides direct mechanistic evidence for the channeling of reaction intermediates, and disruption of this interaction would have important implications on the control of flux in central carbon metabolism.
authors.affiliation: Department of Chemical Engineering, Columbia University , New York, New York 10027, United States.
authors.forename: Beyza
authors.initials: B
authors.lastname: Bulutoglu
authors.affiliation: Department of Chemical Engineering, Columbia University , New York, New York 10027, United States.
authors.forename: Kristen E
authors.initials: KE
authors.lastname: Garcia
authors.affiliation: Department of Chemistry, The University of Utah , Salt Lake City, Utah 84112, United States.|Institute of Chemistry, Chinese Academy of Science , Beijing, China.
authors.forename: Fei
authors.initials: F
authors.lastname: Wu
authors.affiliation: Department of Chemistry, The University of Utah , Salt Lake City, Utah 84112, United States.
authors.forename: Shelley D
authors.initials: SD
authors.lastname: Minteer
authors.affiliation: Department of Chemical Engineering, Columbia University , New York, New York 10027, United States.
authors.forename: Scott
authors.initials: S
authors.lastname: Banta
mesh_terms: D000154:Aconitate Hydratase / Q000737:chemistry / Q000378:metabolism*; D001120:Arginine / Q000235:genetics; D002384:Catalysis; D002950:Citrate (si)-Synthase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002952:Citric Acid Cycle; D007700:Kinetics; D008291:Malate Dehydrogenase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D055672:Static Electricity; D013379:Substrate Specificity
chemical_list: D011994:Recombinant Proteins; D001120:Arginine; D008291:Malate Dehydrogenase; D002950:Citrate (si)-Synthase; D000154:Aconitate Hydratase