title: Effect of the microenvironment on the kinetic properties of immobilized enzymes.
abstract: A new immobilization method was developed in order to perform a systematic study of the influence of the microenvironment on the properties of immobilized enzymes. The enzyme, alcohol dehydrogenase, was first activated, then polypeptide arms of known composition were quantitatively grafted and finally the enzyme was covalently immobilized by co-polymerization of the activated ends of the peptide arms with acrylamide monomers. In this way, the polypeptide linker arms fully determine the properties of the microcavity of the gel in which the enzyme is immobilized by multipoint covalent linkages. The activation energy of the reaction was determined for different microenvironments, in solution as well as after immobilization. Kinetic parameters were also calculated and a new kinetic model was developed, allowing a correction for the diffusional restrictions. The results show that the diffusional restrictions on one hand, and the nature of the microenvironment on the other hand, interact in a dynamic way with the enzyme to determine its properties. Another key point to understanding the changes in the properties of the immobilized enzyme is to consider these proteins as dynamic structures, interacting physically and chemically with their microenvironment.
authors.affiliation: Unité de Biochimie Cellulaire, Facultés Universitaires de Namur, Belgium.
authors.forename: V
authors.initials: V
authors.lastname: Bille
authors.forename: D
authors.initials: D
authors.lastname: Plainchamp
authors.forename: S
authors.initials: S
authors.lastname: Lavielle
authors.forename: G
authors.initials: G
authors.lastname: Chassaing
authors.forename: J
authors.initials: J
authors.lastname: Remacle
mesh_terms: D000426:Alcohol Dehydrogenase / Q000032:analysis*; D055598:Chemical Phenomena; D002621:Chemistry; D004789:Enzyme Activation / Q000187:drug effects; D004800:Enzymes, Immobilized; D007700:Kinetics; D008433:Mathematics; D008962:Models, Theoretical; D015394:Molecular Structure; D010455:Peptides / Q000494:pharmacology; D011487:Protein Conformation; D013388:Succinimides / Q000494:pharmacology
chemical_list: D004800:Enzymes, Immobilized; D010455:Peptides; D013388:Succinimides; C018151:N-succinimidyl 3-(2-pyridyldithio)propionate; D000426:Alcohol Dehydrogenase