title: Investigation of Horseradish Peroxidase Kinetics in an "Organelle-Like" Environment.
abstract: In order to mimic cell organelles, artificial nanoreactors have been investigated based on polymeric vesicles with reconstituted channel proteins (outer membrane protein F) and coencapsulated enzymes horseradish peroxidase (HRP) along with a crowding agent (Ficoll or polyethylene glycol) inside the cavity. Importantly, the presence of macromolecules has a strong impact on the enzyme kinetics, but no influence on the integrity of vesicles up to certain concentrations. This particular design allows for the first time the determination of HRP kinetics inside nanoreactors with crowded milieu. The values of the Michaelis-Menten constant (Km) measured for HRP in a confined space (encapsulated in nanoreactors) in the absence of macromolecules are ≈50% lower than in free conditions, and the presence of a crowding agent results in a further pronounced decrease. These results clearly suggest that activities of enzymes in confined spaces can be tuned by varying the concentrations of crowding compounds. The present investigation represents an advance in nanoreactor design by considering the influence of environmental factors on enzymatic performance, and it demonstrates that both encapsulation and the presence of a crowding environment increase the enzyme-substrate affinity.
authors.affiliation: Department of Chemistry, University of Basel, Klingelbergstrasse 80, 4056, Basel, Switzerland.
authors.forename: Patric
authors.initials: P
authors.lastname: Baumann
authors.affiliation: Department of Chemistry, University of Basel, Klingelbergstrasse 80, 4056, Basel, Switzerland.
authors.forename: Mariana
authors.initials: M
authors.lastname: Spulber
authors.affiliation: Department of Chemistry, University of Basel, Klingelbergstrasse 80, 4056, Basel, Switzerland.
authors.forename: Ozana
authors.initials: O
authors.lastname: Fischer
authors.affiliation: Department of Chemistry, University of Basel, Klingelbergstrasse 80, 4056, Basel, Switzerland.
authors.forename: Anja
authors.initials: A
authors.lastname: Car
authors.affiliation: Department of Chemistry, University of Basel, Klingelbergstrasse 80, 4056, Basel, Switzerland.
authors.forename: Wolfgang
authors.initials: W
authors.lastname: Meier
mesh_terms: D004800:Enzymes, Immobilized / Q000737:chemistry* / Q000378:metabolism*; D006735:Horseradish Peroxidase / Q000737:chemistry* / Q000378:metabolism*; D007700:Kinetics
chemical_list: D004800:Enzymes, Immobilized; D006735:Horseradish Peroxidase
keywords: encapsulation efficiency; enzyme kinetics; macromolecular crowding; nanoreactors