Subtle differences in dissociation rates of interactions between destabilized human carbonic anhydrase II mutants and immobilized benzenesulfonamide inhibitors probed by a surface plasmon resonance biosensor

The development of commercial biosensors based on surface plasmon resonance has made possible careful characterization of biomolecular interactions. H ere, a set of destabilized human carbonic anhydrase II (HCA II) mutants was investigated with respect to their interaction kinetics with two different immobilized benzenesulfonamide inhibitors. Point mutations were located di stantly from the active site, and the destabilization energies were up to 2 3 kJ/mol. The dissociation rate of wild-type HCA II, as determined from the binding to the inhibitor with higher affinity, was 0.019 s(-1). For the mu tants, dissociation rates were faster (0.022-0.025 s(-1)), and a correlatio n between faster dissociation and a high degree of destabilization was obse rved. We interpreted these results in terms of increased dynamics of the te rtiary structures of the mutants. This interpretation was supported by entr opy determinations, showing that the entropy of the native structure signif icantly increased upon destabilization of the protein molecule. Our finding s demonstrate the applicability of modern biosensor technology in the study of subtle details in molecular interaction mechanisms, such as the long-ra nge effect of point mutations on interaction kinetics. (C) 2001 Academic Pr ess.