We study the hyperactivation of α-chymotrypsin (α-ChT) here, using acrylate-derived PCEA polymer. Enzyme activity assays revealed a pronounced enzyme hyperactivation capacity being superior to widespread PAA polymers. In a combined experimental and computational study, we investigate α-ChT/polymer interactions to elucidate the hyperactivation mechanism.

Abstract

We study the hyperactivation of α-chymotrypsin (α-ChT) using the acrylate polymer poly(2-carboxyethyl) acrylate (PCEA) in comparison to the commonly used poly(acrylic acid) (PAA). The polymers are added during the enzymatic cleavage reaction of the substrate N-glycyl-L-phenylalanine p-nitroanilide (GPNA). Enzyme activity assays reveal a pronounced enzyme hyperactivation capacity of PCEA, which reaches up to 950 % activity enhancement, and is significantly enhanced to PAA (revealing an activity enhancement of approx. 450 %). In a combined experimental and computational study, we investigate α-ChT/polymer interactions to elucidate the hyperactivation mechanism of the enzyme. Isothermal titration calorimetry reveals a pronounced complexation between the polymer and the enzyme. Docking simulations reveal that binding of polymers significantly improves the binding affinity of GPNA to α-ChT. Notably, a higher binding affinity is found for the α-ChT/PCEA compared to the α-ChT/PAA complex. Further molecular dynamics (MD) simulations reveal changes in the size of the active site in the enzyme/polymer complexes, with PCEA inducing a more pronounced alteration compared to PAA, facilitating an easier access for the substrate to the active site of α-ChT.