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.

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“…The vinyl sulfone terminated DMA-AZAAm copolymer was also successfully conjugated to a cysteine-modified mutant of the enzyme endoglucanase EG 12A. 10 Under irradiation with visible light, the soluble polymer chain did not alter substrate binding, and the catalytic activity of the polymer-enzyme conjugate was similar to that of the unmodified control enzyme. However, upon UV irradiation, the DMAAZAAm copolymer collapsed and blocked substrate access; this was reflected in a strong decrease in the catalytic activity.…”

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“…This suggested that the orientation of the enzyme active site could be controlled in the particle such that substrate binding was allowed or blocked, depending on the orientation of the polymer relative to the active site (Figure 16.3). This application was demonstrated with both thermal and photo-responsive polymers that served as molecular switches to control the activity of the commercially important enzyme, endoglucanase 12A [2,33,34].…”