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

Abstract

Recently, a homozygous deletion mutation of one of the two contiguous Lys9 ⁄ Lys10 residues in the A-chain of a-thrombin (DK9) was identified in patients with severe prothrombin deficiency and hemorrhagic diathesis [1,2].Compared with the wild-type (WT) form, the specificity constants of hydrolysis by DK9 of the synthetic substrate d-Phe-Pip-Arg-pNA and fibrinopeptide A were found to be 18-and 60-fold lower, respectively. The catalytic competence of the natural thrombin mutant with deletion of the Lys9 residue in the A-chain (DK9) 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-a-(2-naphthylsulfonyl-glycyl)-4-amidinophenylalanine-piperidine (a-NAPAP) to the wild-type (WT) and DK9 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 pK a value of the first ionizable group, assigned to the catalytic His57 residue, was found to be 7.5 and 6.9 in ligand-free DK9 and WT thrombin, respectively. Urea-induced denaturation studies showed higher instability of the DK9 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.Abbreviations a-NAPAP, N-a-(2-naphthylsulfonyl-glycyl)-4-amidinophenylalanine-piperidine; Bis-Tris, (2-hydroxyethyl)iminotris(hydroxymethyl)methane;

Role of the A chain in thrombin function

“…Notably, the perturbed structure produced by modeling contains features documented in the X-ray crystal structure of the inactive form of thrombin E* [25]. Recent measurements of the pKa of the catalytic His57 further support a direct effect on active site residues [10]. Our study shows that the K9A and K10A mutants have functional properties comparable to those of wild type.…”

“…Deletion of Lys9 in the A chain is associated with a bleeding phenotype [8]. The thrombin mutant ΔK9 shows defects in the activation of its zymogen form [9], but the mature enzyme features impaired Na + binding and significantly reduced activity toward a chromogenic substrate, fibrinogen, PAR1 and protein C [9] due to long-range perturbation of the active site moiety and catalytic triad [9,10]. These findings underscore the importance of the A chain in thrombin function and warrant a thorough analysis in terms of mutagenesis.…”

Thrombin

“…Other naturally mutations, like deletion of K9 or K10 (Akhavan et al, 2000), are also associated with severe bleeding. Interestingly, the defect causes impaired fibrinogen and PAR1 cleavage, reduced response to Na + activation (De Cristofaro et al, 2004, and long-range perturbation of active site residues (De Cristofaro et al, 2006). The A chain is rich in charged residues that make polar interactions with partners of the B chain.…”

Thrombin A-Chain: Activation Remnant or Allosteric Effector?

“…The authors suggested that hypoprothrombinemia is caused by the incomplete folding of the A-chain, which is then unable to stabilize the B-chain structure. Two recent studies on the Lys301 deletion prothrombin mutant have suggested that A-chain structure affects the conformation and catalytic properties of thrombin through long range allosteric effects on the active site and insertion loops [25, 26]. Interestingly, a recent crystal structure of thrombin complexed with sulfo-hirudin found that Lys301 participates in a divalent metal binding site between the heavy and light chains of thrombin [27].…”