ABSTRACT
This account presents a general method for the construction of polymeric surface binders for digestion enzymes. Two prominent parts, namely, the modification of the copolymer composition and the screening assay for the most powerful inhibitors are both amenable to parallelization. The concept hinges on the appropriate selection of amino-acid-selective comonomers, their free radical copolymerization, and subsequent screening of the resulting copolymer library for efficient enzyme inhibition. A microscale synthetic procedure for the copolymerization process was developed, which produces water-soluble affinity polymers that can be stored for years at room temperature. Initial parallel screening was conducted in standard enzyme assays to identify polymeric inhibitors, which were subsequently subjected to determination of IC50 values for their target enzyme. For all digestion enzymes, except elastase, a number of polymer inhibitors were found, some of which were selective toward one or two protein targets. Since the key monomers of the best inhibitors bind to amino acid residues in the direct vicinity of the active site, we conclude that efficient coverage of the immediate environment by the copolymers is critical. Strong interference with enzymatic activity is brought about by blocking the substrate access and product exit to and from the active site.
Subject(s)
Benzamidines/chemistry , Diphosphonates/chemistry , Enzyme Inhibitors/chemistry , Pancreatic Elastase/chemistry , Polymers/chemistry , Serine Proteases/chemistry , Alanine/chemistry , Aspartic Acid/chemistry , Benzamidines/chemical synthesis , Catalytic Domain , Diphosphonates/chemical synthesis , Enzyme Assays , Enzyme Inhibitors/chemical synthesis , Glutamic Acid/chemistry , Humans , Kinetics , Pancreatic Elastase/antagonists & inhibitors , Polymerization , Polymers/chemical synthesis , Protein BindingABSTRACT
A new concept for the external control of protein activity is presented and demonstrated on the example of an artificial Lysozyme switch. Radical copolymerization of selected methacrylamide-based comonomer units tailored for amino acid residues surrounding the active site furnishes polymeric protein hosts that are able to inhibit enzymatic activity in a highly efficient dose-dependent manner (IC50 approximately 1.0 equiv approximately 0.7 microM). All binding site types on the polymer work cooperatively, using electrostatic attraction, hydrophobic forces, and substrate mimicry. In a native gel electrophoresis, the well-defined 2:1 complex (polymer/protein) migrates to the anode. Even at 250 mM NaCl, a 10-fold polymer excess is able to shut down bacterial cell wall degradation completely. A kinetic investigation points to a competitive mechanism (Lineweaver-Burk plots). CD spectra of pure Lysozyme and its polymer complex are indistinguishable; together with a total lack of preincubation time for maximum inhibition, experimental evidence is thus produced for a preserved tertiary enzyme structure-no denaturation occurs. Addition of the superior complexing agent polyarginine to the enzyme-polymer complex mildly detaches the inhibitor from the protein surface and leads to 90% recovery of enzymatic activity. Thus, Lysozyme could be turned off, on, and off again by consecutive addition of the polymeric inhibitor, polyarginine, and polymer again.
