Hybrid hydrogels are hydrogels synthesized by two distinct components, synthetic polymers and biomacromolecules such as proteins, peptides or DNA. Due to their smart properties, hybrid hydrogels are widely used as biomaterials responsive to external stimuli and drug delivery systems. This study provides the first example of dynamic hybrid hydrogels able to translate enzyme-substrate recognition into mechanical motion. Adenylate kinase, which catalyzes the conversion of one mole of ATP and one mole of AMP to two moles of ADP with the help of magnesium ions, was found to have controlled conformational changes upon the binding of substrates (ATP, AMP, ADP) or inhibitors (such as the two-substrate mimicking inhibitor, Ap5A).

(C77S, V169C, A55C)-triple mutant adenylate kinase containing two thiol groups at positions with the largest spatial displacement was expressed from plasmid prepared by site-specific mutagenesis, purified by affinity chromatography and gel filtration and identified by electro-spray ionization mass spectrometry. Hybrid enzyme-based hydrogels were prepared by thiol-maleimide conjugation reactions of mutated adenylate kinase with pendant maleimide groups of N-(2-hydroxypropyl)methacylamide (HPMA) copolymer or maleimido-terminated star-shaped poly(ethylene glycol) (PEG). The de-swelling ratios of the hybrid hydrogels when exposed to buffer containing substrate were measured, and the results were in agreement with the conformational change of adenylate kinase from “open†state to “closed†state upon substrate binding. A hybrid hydrogel prepared with fluorescence-labeled adenylate kinase emitted fluorescence after extensive washing, proving the covalent attachment of adenylate kinase to the three-dimensional network. The equilibrium swelling ratios were determined gravimetrically and the morphology of the hydrogels exhibited porous interconnected structures on scanning electron microscopy. The results obtained suggest that these hydrogels have application potential in biosensor technology and microfluidic systems.