tailieunhanh - Báo cáo khoa học: 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

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) simula-tions in explicit water solvent. | ềFEBS Journal 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 - I l-s o. . r 1 A I Z- .-2 -IIAII 3. l -Vll3 l 1-3 Raimondo De Cristofaro Andrea Carotti Sepideh Akhavan Roberta Palla Flora Peyvandi Cosimo Altomare2 and Pier Mannuccio Mannucci3 1 Haemostasis Research Centre Institute of InternalMedicine and Geriatrics Catholic University Schoolof Medicine Rome Italy 2 Department of PharmaceuticalChemistry University of Bari Italy 3 Angelo Bianchi Bonomi Hemophilia and Thrombosis Center and Fondazione Luigi Villa IRCCS Maggiore HospitalUniversity of Milan Italy Keywords allostery molecular dynamics pKa values stability thrombin Correspondence R. De Cristofaro Haemostasis Research Centre Institute of InternalMedicine and Geriatrics Catholic University Schoolof Medicine Largo F. Vito 1 00168 Rome Italy Fax 39 6 30 155915 Tel 39 6 30 154 438 E-mail rdecristofaro@ C. Altomare Department of Pharmaceutical Chemistry University of Bari Via E. Orabona 4 70125 Bari Italy Fax 39 80 544 2230 Tel 39 80 544 2781 E-mail altomare@ Present address INSERM E0348 Faculte Xavier Bichat University Paris 7 France Received 2 September 2005 revised 12 October 2005 accepted 7 November 2005 doi 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