title: Colocalization and Sequential Enzyme Activity in Aqueous Biphasic Systems: Experiments and Modeling.
abstract: Subcellular compartmentalization of biomolecules and their reactions is common in biology and provides a general strategy for improving and/or controlling kinetics in metabolic pathways that contain multiple sequential enzymes. Enzymes can be colocalized in multiprotein complexes, on scaffolds or inside subcellular organelles. Liquid organelles formed by intracellular phase coexistence could provide an additional means of sequential enzyme colocalization. Here we use experiment and computation to explore the kinetic consequences of sequential enzyme compartmentalization into model liquid organelles in a crowded polymer solution. Two proteins of the de novo purine biosynthesis pathway, ASL (adenylosuccinate lyase, Step 8) and ATIC (5-aminoimidazole-4-carboxamide ribonucleotide transformylase/inosine monophosphate cyclohydrolase, Steps 9 and 10), were studied in a polyethylene glycol/dextran aqueous two-phase system. Dextran-rich phase droplets served as model liquid compartments for enzyme colocalization. In this system, which lacks any specific binding interactions between the phase-forming polymers and the enzymes, we did not observe significant rate enhancements from colocalization for the overall reaction under our experimental conditions. The experimental results were used to adapt a mathematical model to quantitatively describe the kinetics. The mathematical model was then used to explore additional, experimentally inaccessible conditions to predict when increased local concentrations of enzymes and substrates can (or cannot) be expected to yield increased rates of product formation. Our findings indicate that colocalization within these simplified model liquid organelles can lead to enhanced metabolic rates under some conditions, but that very strong partitioning into the phase that serves as the compartment is necessary. In vivo, this could be provided by specific binding affinities between components of the liquid compartment and the molecules to be localized within it.
authors.affiliation: Department of Chemistry, The Pennsylvania State University, University Park, Pennsylvania.
authors.forename: Bradley W
authors.initials: BW
authors.lastname: Davis
authors.affiliation: Department of Chemistry, The Pennsylvania State University, University Park, Pennsylvania.
authors.forename: William M
authors.initials: WM
authors.lastname: Aumiller
authors.affiliation: Department of Chemical Engineering, The Pennsylvania State University, University Park, Pennsylvania.
authors.forename: Negar
authors.initials: N
authors.lastname: Hashemian
authors.affiliation: Department of Chemistry, The Pennsylvania State University, University Park, Pennsylvania.
authors.forename: Songon
authors.initials: S
authors.lastname: An
authors.affiliation: Department of Chemical Engineering, The Pennsylvania State University, University Park, Pennsylvania. Electronic address: armaou@engr.psu.edu.
authors.forename: Antonios
authors.initials: A
authors.lastname: Armaou
authors.affiliation: Department of Chemistry, The Pennsylvania State University, University Park, Pennsylvania. Electronic address: keating@chem.psu.edu.
authors.forename: Christine D
authors.initials: CD
authors.lastname: Keating
mesh_terms: D000264:Adenylosuccinate Lyase / Q000737:chemistry / Q000378:metabolism*; D002451:Cell Compartmentation; D006801:Humans; D019877:Hydroxymethyl and Formyl Transferases / Q000737:chemistry / Q000378:metabolism*; D008081:Liposomes / Q000737:chemistry; D008954:Models, Biological; D009097:Multienzyme Complexes / Q000737:chemistry / Q000378:metabolism*; D009709:Nucleotide Deaminases / Q000737:chemistry / Q000378:metabolism*
chemical_list: D008081:Liposomes; D009097:Multienzyme Complexes; C113120:inosine monophosphate synthase; D019877:Hydroxymethyl and Formyl Transferases; D009709:Nucleotide Deaminases; D000264:Adenylosuccinate Lyase