Perturbation of pKa values of catalytic residues in the NATURAL thrombin : mutant with deletion of Lys9 in the A-chain

The catalytic competence of the natural thrombin mutant with deletion of Lys9 residue in the A-chain (K9) had been found severely impaired, most likely due to modifications of the 60-loop conformation and catalytic triad geometry, as highlighted by long molecular dynamics (MD) simulations in fully explicit water solvent. In this study, the protonation state of active site’s residues in wild type (WT) and K9 thrombins was investigated analysing the pH effect on the enzyme’s amidase activity and binding of the inhibitor N--[2-naphthylsulfonyl-glycyl]-4-amidinophenylalanine-piperidide (-NAPAP). Two ionisable groups were found to affect similarly the activity of both thrombins. The pKa value of the first ionisable group, assigned to the His57 residue, was found to be 7.5 and 6.9 in the ligand-free K9 and WT thrombins, respectively. Likewise, the pKa value of the N-terminus group (NTIle) in K9 mutant underwent a moderate but significant increase over the WT pKa value (8.9 vs.8.5). Theoretical methods for calculating pKa values of ionisable amino acid residues, when applied to MD snapshot structures calculated in the first part of MD trajectories, where more open proteins’ structures simulate thrombins in their procoagulant conformations, yielded pKa values for His57 (5.6 and 7.4 for the ligand-free WT and K9 thrombins, respectively), but also for NTIle, in qualitative agreement with the experimental ones. Taken together, these findings contribute to increase our understanding of the perturbations triggered by Lys9 deletion, which may directly and/or allosterically affect the protonation state of the catalytic residues involved in both ligand binding and proteolytic activity