Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.4.21.86 (clotting enzyme)
 176 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Hirudin is the most potent and specific inhibitor of the blood-clotting enzyme thrombin so far known. Several hirudin variants were isolated mostly from Hirudo medicinalis and shown to be polypeptide chains of approximately 7 kDa with three internal disulfide bridges. In this study, limited proteolysis has been used to probe aspects of the structure and dynamics of a hirudin variant HM2 isolated from Hirudinaria manillensis. Proteolysis of the polypeptide chain of 64-amino-acid residues of hirudin HM2 by protease from Staphylococcus aureus V8, trypsin, thermolysin and subtilisin occurs at region 41-49 of the chain. The N-terminal fragments 1-41 and 1-47 were isolated to homogeneity and shown to maintain inhibitory action on thrombin, though much lower than the intact protein. The results were interpreted on the basis of a proposed three-dimensional structure of hirudin HM2 deduced by protein modelling the known structure of hirudin variant HV1 from Hirudo medicinalis (75% sequence similarity between HM2 and HV1). Both proteolysis experiments and protein modelling provide evidence for the existence in hirudin HM2 of a N-terminal well-structured domain (core) and a C-terminal flexible polypeptide segment. Determination of the accessible surface area of the three-dimensional model of hirudin HM2 showed that the sites of preferential cleavages are at the surface of the polypeptide molecule.
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PMID:Probing the structure of hirudin from Hirudinaria manillensis by limited proteolysis. Isolation, characterization and thrombin-inhibitory properties of N-terminal fragments. 800 50

The 64 amino acid hirudin-like peptide HM2 (Hirudinaria manillensis) is one of the agents known to specifically block the blood-clotting enzyme thrombin, and therefore is used as a potential pharmacological tool for the treatment of arterial and venous thrombosis. This peptide and its derivatives provide a new set of probes for studies aimed at elucidating the structural basis of the inhibition of alpha-thrombin. We used 581, 699, and 492 nmr-derived constraints respectively in a protocol employing simulated annealing, followed by restrained molecular dynamics and restrained energy minimization to derive the three-dimensional structures of HM2 and its mutants the HM2 (V + G) and the HM2 (1-47). HM2 consists of a well-defined core region of two double-stranded beta-sheet and a disordered C-terminus. These features are shared by other members of the hirudin family. The same type of folding has also been observed for recombinant hirudins whose structure has been determined in solution by nmr spectroscopy and in the structure of the complex hirudin-thrombin determined by x-ray diffraction. Molecular dynamics (MD) simulation methods were applied in the study of the structural and dynamic fluctuation properties of the hirudin derivatives solvated by 1625 and 1276 water molecules with periodic boundary conditions for HM2 and HM2 (1-47), respectively. Trajectories of 100 and 50 ps for the two unconstrained systems were generated at constant temperature and pressure. Analysis of the MD simulation shows that the structure of the peptide core is fairly rigid and stable in itself while the conformation of the C-terminal tail, which is involved in the inhibitory mechanism of thrombin, fluctuates and appears as a disordered region.
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PMID:NMR solution structure of a novel hirudin variant HM2, N-terminal 1-47 and N64-->V + G mutant. 912 39