The natural mutation by deletion of Lys9 in the thrombin A‐chain affects the pKa value of catalytic residues, the overall enzyme's stability and conformational transitions linked to Na+ binding

Open Access
Publisher Website
Google Scholar
Abstract
The catalytic competence of the natural thrombin mutant with deletion of the Lys9 residue in the A‐chain (ΔK9) was found to be severely impaired, most likely due to modification of the 60‐loop conformation and catalytic triad geometry, as supported by long molecular dynamics (MD) simulations in explicit water solvent. In this study, the pH dependence of the catalytic activity and binding of the low‐molecular mass inhibitor N‐α‐(2‐naphthylsulfonyl‐glycyl)‐4‐amidinophenylalanine‐piperidine (α‐NAPAP) to the wild‐type (WT) and ΔK9 thrombin forms were investigated, along with their overall structural stabilities and conformational properties. Two ionizable groups were found to similarly affect the activity of both thrombins. The pKa value of the first ionizable group, assigned to the catalytic His57 residue, was found to be 7.5 and 6.9 in ligand‐free ΔK9 and WT thrombin, respectively. Urea‐induced denaturation studies showed higher instability of the ΔK9 mutant compared with WT thrombin, and disulfide scrambling experiments proved weakening of the interchain interactions, causing faster release of the reduced A‐chain in the mutant enzyme. The sodium ion binding affinity was not significantly perturbed by Lys9 deletion, although the linked increase in intrinsic fluorescence was lower in the mutant. Essential dynamics (ED) analysis highlighted different conformational properties of the two thrombins in agreement with the experimental conformational stability data. Globally, these findings enhanced our understanding of the perturbations triggered by Lys9 deletion, which reduces the overall stability of the molecule, weakens the A–B interchain interactions, and allosterically perturbs the geometry and protonation state of catalytic residues of the enzyme.