title: Enzyme activity in the crowded milieu.
abstract: The cytosol of a cell is a concentrated milieu of a variety of different molecules, including small molecules (salts and metabolites) and macromolecules such as nucleic acids, polysaccharides, proteins and large macromolecular complexes. Macromolecular crowding in the cytosolic environment is proposed to influence various properties of proteins, including substrate binding affinity and enzymatic activity. Here we chose to use the synthetic crowding agent Ficoll, which is commonly used to mimic cytosolic crowding conditions to study the crowding effect on the catalytic properties of glycolytic enzymes, namely phosphoglycerate kinase, glyceraldehyde 3-phosphate dehydrogenase, and acylphosphatase. We determined the kinetic parameters of these enzymes in the absence and in the presence of the crowding agent. We found that the Michaelis constant, K(m), and the catalytic turnover number, k(cat), of these enzymes are not perturbed by the presence of the crowding agent Ficoll. Our results support earlier findings which suggested that the Michaelis constant of certain enzymes evolved in consonance with the substrate concentration in the cell to allow effective enzyme function in bidirectional pathways. This conclusion is further supported by the analysis of nine other enzymes for which the K(m) values in the presence and absence of crowding agents have been measured.
authors: Department of Biology and Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy, New York, United States of America.
authors: Tobias
authors: T
authors: Vöpel
authors: George I
authors: GI
authors: Makhatadze
mesh_terms: D017766:Acid Anhydride Hydrolases / Q000378:metabolism*; D003600:Cytosol / Q000201:enzymology*; D005362:Ficoll / Q000494:pharmacology; D024601:Glyceraldehyde 3-Phosphate Dehydrogenase (NADP+) / Q000378:metabolism*; D007700:Kinetics; D010735:Phosphoglycerate Kinase / Q000378:metabolism*; D000097024:Acylphosphatase
chemical_list: D017766:Acid Anhydride Hydrolases; D005362:Ficoll; D024601:Glyceraldehyde 3-Phosphate Dehydrogenase (NADP+); D010735:Phosphoglycerate Kinase; D000097024:Acylphosphatase