Control of cascade reaction by proximity of enzymes.

Abstract

A proximity of enzymes into a cytoplasm causes efficient cascade reactions of enzymes. Therefore, efficiencies of enzyme reactions in vitro will be improved by a local increase in concentration of enzyme. In a previous study, two different types of enzymes were placed in close proximity using synthetic polymer. However, it is difficult to reversibly control proximity and dispersion of enzymes. In this study, we attempt to closely assemble three enzymes using 3-way junction DNA in order to create an efficient and controllable bioreactor emulating the proximity of enzymes into a cytoplasm. The methods for constructions of three-enzyme complexes show the following steps. First, we designed single stranded DNAs (ssDNAs) which can form the triply-branched structure by a hybridization reaction. The ssDNA with amino group at terminal was conjugated to NHS ester-PEG4-maleimide. Next, a sulfhydryl group of primary amines of three enzymes (horseradish peroxidase, glucose oxidase and β-galactosidase) was reacted with maleimide of ssDNA-PEG4-maleimide. To obtain ssDNA-PEG4-Protein, three types of thiolated enzymes were conjugated with three different ssDNA-PEG4-maleimide, correspondingly. The products were purified and characterized by native PAGE and UV-Vis spectroscopy. The absorbance intensity of UV-Vis spectra of the purified ssDNA-PEG4-Protein at 260 nm for the ssDNA depended on the number of ssDNAs in a protein. Finally, three-enzyme complexes were assembled. The enzyme activity of three-enzyme complexes was compared with that of the free enzyme mixture. The cascade reaction is involving β-Gal-catalyzed hydrolysis of lactose, GOx-catalyzed oxidation of glucose to gluconolactone and hydrogen peroxide, and HRP-catalyzed oxidation of Amplex Red to resorufin with hydrogen peroxide. Assembling of three-enzyme complexes using 3-way junction DNA enhanced the cascade reaction activity under a dilute condition compared with the free enzymes. Therefore, this enzyme assembly system will contribute to emulations efficient reactions into the biological cells.