title: Two-Site Internally Cooperative Mechanism for Enzyme Kinetics in a Hydrogel Forming Recombinant Protein.
abstract: A recombinant protein, ATCTA, consisting of three domains, α-helix (A), thrombin cleavage site (T), and water-soluble coil (C), forms hydrogels via the self-association of its flanking α-helices into tetrameric bundles, which act as cross-links for the hydrogel network. In the presence of thrombin, the hydrogel degrades due to the thrombin cleavage sites. To better understand the proteolysis reaction in ATCTA, we performed a series of kinetic experiments on the proteins ATC, CTA, CTATC, and ATCTA. The KM and kcat of ATC and CTA were determined to be 88 ± 5 μM and 6.4 ± 0.1 s(-1) and 91 ± 9 μM and 6.1 ± 0.1 s(-1), respectively. Using these kinetic parameters, a model based on a two-site internally cooperative mechanism was developed to describe the kinetics of proteins containing two cleavage sites. This model was then validated by comparing predicted results with kinetic data from the proteolysis of ATCTA.
authors.affiliation: Anderson University Department of Chemistry and Physics, 1100 East Fifth Street Anderson, Indiana 46012, United States.
authors.forename: Nicholas A
authors.initials: NA
authors.lastname: Zehner
authors.affiliation: Anderson University Department of Chemistry and Physics, 1100 East Fifth Street Anderson, Indiana 46012, United States.
authors.forename: Scott M
authors.initials: SM
authors.lastname: Dietrick
authors.affiliation: Division of Chemistry and Chemical Engineering, California Institute of Technology , 1200 E. California Blvd., Pasadena, California 91125 United States.
authors.forename: David A
authors.initials: DA
authors.lastname: Tirrell
authors.affiliation: Anderson University Department of Chemistry and Physics, 1100 East Fifth Street Anderson, Indiana 46012, United States.
authors.forename: Scott B
authors.initials: SB
authors.lastname: Kennedy
mesh_terms: D004798:Enzymes / Q000737:chemistry*; D020100:Hydrogels / Q000737:chemistry*; D008958:Models, Molecular; D017433:Protein Structure, Secondary; D059748:Proteolysis; D011994:Recombinant Proteins / Q000737:chemistry* / Q000235:genetics; D013917:Thrombin / Q000737:chemistry
chemical_list: D004798:Enzymes; D020100:Hydrogels; D011994:Recombinant Proteins; D013917:Thrombin