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

Raimondo De Cristofaro, Andrea Carotti, Sepideh Akhavan, Roberta Palla, Flora Peyvandi, Cosimo Altomare, Pier Mannuccio Mannucci

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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.

Original languageEnglish
Pages (from-to)159-169
Number of pages11
JournalFEBS Journal
Volume273
Issue number1
DOIs
Publication statusPublished - Jan 2006

Fingerprint

Enzyme Stability
Sequence Deletion
Thrombin
Enzymes
Denaturation
Geometry
Protonation
Molecular mass
Molecular Dynamics Simulation
Disulfides
Mental Competency
Dynamic analysis
Conformations
Urea
Molecular dynamics
Catalyst activity
Fluorescence
Sodium
Ions
Ligands

Keywords

  • Allostery
  • Molecular dynamics
  • pK values
  • Stability
  • Thrombin

ASJC Scopus subject areas

  • Biochemistry

Cite this

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title = "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",
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.",
keywords = "Allostery, Molecular dynamics, pK values, Stability, Thrombin",
author = "{De Cristofaro}, Raimondo and Andrea Carotti and Sepideh Akhavan and Roberta Palla and Flora Peyvandi and Cosimo Altomare and Mannucci, {Pier Mannuccio}",
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T1 - 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

AU - De Cristofaro, Raimondo

AU - Carotti, Andrea

AU - Akhavan, Sepideh

AU - Palla, Roberta

AU - Peyvandi, Flora

AU - Altomare, Cosimo

AU - Mannucci, Pier Mannuccio

PY - 2006/1

Y1 - 2006/1

N2 - 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.

AB - 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.

KW - Allostery

KW - Molecular dynamics

KW - pK values

KW - Stability

KW - Thrombin

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U2 - 10.1111/j.1742-4658.2005.05052.x

DO - 10.1111/j.1742-4658.2005.05052.x

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SN - 1742-464X

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