Functional mapping of charged residues of the 82-116 sequence in factor Xa: evidence that lysine 96 is a factor Va independent recognition site for prothrombin in the prothrombinase complex.
Research article published in Biochemistry (2005)
Abstract
It has been hypothesized that two antiparallel structures comprised of residues 82-91 and 102-116 in factor Xa (fXa) may harbor a factor Va- (fVa-) dependent prothrombin recognition site in the prothrombinase complex. There are 11 charged residues in the 82-116 loop of human fXa (Glu-84, Glu-86, Lys-90, Arg-93, Lys-96, Glu-97, Asp-100, Asp-102, Arg-107, Lys-109, and Arg-115). With the exception of Glu-84, which did not express, and Asp-102, which is a catalytic residue, we expressed the Ala substitution mutants of all other residues and evaluated their proteolytic and amidolytic activities in both the absence and presence of fVa. K96A and K109A activated prothrombin with 5-10-fold impaired catalytic efficiency in the absence of fVa. All mutants, however, exhibited normal activity toward the substrate in the presence of fVa. K109A also exhibited impaired amidolytic activity and affinity for Na(+); however, both fVa and higher Na(+) restored the catalytic defect caused by the mutation. Analysis of the X-ray crystal structure of fXa indicated that Glu-84 may interact by a salt bridge with Lys-109, explaining the lack of expression of E84A and the lower activity of K109A in the absence of fVa. These results suggest that none of the residues under study is a fVa-dependent recognition site for prothrombin in the prothrombinase complex; however, Lys-96 is a recognition site for the substrate independent of the cofactor. Moreover, the 82-116 loop is energetically linked to fVa and Na(+) binding sites of the protease.
Abstract sourced from PubMed (NCBI) for the cited record. See the original publication for the authoritative version.
Resumen
It has been hypothesized that two antiparallel structures comprised of residues 82-91 and 102-116 in factor Xa (fXa) may harbor a factor Va- (fVa-) dependent prothrombin recognition site in the prothrombinase complex. There are 11 charged residues in the 82-116 loop of human fXa (Glu-84, Glu-86,...
Por qué esto importa para la hirudoterapia
Este estudio empleó mutagénesis por sustitución de alanina para investigar los residuos cargados en el bucle 82-116 del factor Xa humano, probando si conforman un sitio de reconocimiento de protrombina dependiente del factor Va en el complejo protrombinasa. Los autores hallaron que los mutantes K96A y K109A mostraron una eficiencia catalítica para la activación de protrombina alterada en un factor de 5 a 10 en ausencia del factor Va, aunque todos los mutantes exhibieron actividad normal en su presencia, concluyendo que la Lys-96 funciona como un sitio de reconocimiento del sustrato independiente del cofactor, mientras que ninguno de los residuos analizados cumple una función dependiente del factor Va. Este trabajo resulta relevante para la hirudoterapia porque el factor Xa genera trombina —la enzima directamente inhibida por la hirudina derivada de la sanguijuela— y dilucidar el ensamblaje de la protrombinasa avanza en la comprensión de la cascada de coagulación que los anticoagulantes de la sanguijuela medicinal se utilizan para modular. La limitación clave es que este estudio de bioquímica básica no involucra sustancias derivadas de sanguijuelas, y su conexión con la terapia clínica con sanguijuelas es completamente indirecta.
Citación
Functional mapping of charged residues of the 82-116 sequence in factor Xa: evidence that lysine 96 is a factor Va independent recognition site for prothrombin in the prothrombinase complex.
Manithody et al. · Biochemistry, 2005
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Añadido a la biblioteca ASH: May 28, 2026 · Última actualización del sitio: 18 de junio de 2026