title: Temperature-induced switching of enzyme activity with smart polymer-enzyme conjugates.
abstract: A method for thermally induced switching of enzyme activity has been developed, based on the site-directed conjugation of end-reactive temperature-responsive polymers to a unique cysteine (Cys) residue positioned near the enzyme active site. The reversible temperature-induced collapse of N,N-dimethylacrylamide (DMA)/N-4-phenylazo-phenylacrylamide (AZAAm) copolymers (DMAAm) has been used as a molecular switch to control the catalytic activity of endoglucanase 12A (EG 12A). The polymer was conjugated to the EG 12A site-directed mutant N55C, directly adjacent to the cellulose binding cleft, and to the S25C mutant, where the conjugation site is more distant. The N55C conjugate displayed a larger activity shutoff efficiency in the collapsed polymer state than the S25C conjugate. Increasing the polymer molecular weight was also shown to increase the shutoff efficiency of the switch. Related to these effects of conjugation site and polymer size, the switching efficiency was found to be strongly dependent on substrate size. With a small substrate, o-nitrophenyl-beta-d-cellobioside (ONPC), there was minimal blocking of enzyme activity when the polymer was in the expanded state. With a large substrate, hydroxyethyl cellulose (HEC), there was a large reduction of enzyme activity in the polymer expanded state, even with relatively small polymer chains, and a further reduction when the polymer was collapsed. Similar general trends for the interactive effects of conjugation site, polymer size, and substrate size were observed for immobilized conjugates. Kinetic studies demonstrated that the switching activity was due to the blocking of substrate association by the collapsed polymers. These investigations provide mechanistic insight that can be utilized to design molecular switches for a variety of stimuli-responsive polymer-protein conjugates.
authors.affiliation: Department of Bioengineering, University of Washington, Seattle, Washington 98195, USA.
authors.forename: Tsuyoshi
authors.initials: T
authors.lastname: Shimoboji
authors.forename: Edmund
authors.initials: E
authors.lastname: Larenas
authors.forename: Tim
authors.initials: T
authors.lastname: Fowler
authors.forename: Allan S
authors.initials: AS
authors.lastname: Hoffman
authors.forename: Patrick S
authors.initials: PS
authors.lastname: Stayton
mesh_terms: D002384:Catalysis; D004798:Enzymes / Q000138:chemical synthesis / Q000378:metabolism*; D008958:Models, Molecular; D011108:Polymers / Q000138:chemical synthesis / Q000378:metabolism*; D013696:Temperature
chemical_list: D004798:Enzymes; D011108:Polymers