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.
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.
Manithody
Chandrashekhara
C
Edward A. Doisy Department of Biochemistry and Molecular Biology, St. Louis University School of Medicine, St. Louis, Missouri 63104, USA.
Rezaie
Alireza R
AR
D000409:Alanine / Q000235:genetics; D000577:Amides / Q000737:chemistry; D000991:Antithrombins / Q000737:chemistry; D002384:Catalysis; D002460:Cell Line; D015943:Factor Va / Q000737:chemistry* / Q000378:metabolism; D015951:Factor Xa / Q000737:chemistry* / Q000235:genetics; D065427:Factor Xa Inhibitors; D006801:Humans; D006868:Hydrolysis; D008239:Lysine / Q000235:genetics / Q000378:metabolism*; D010446:Peptide Fragments / Q000737:chemistry* / Q000235:genetics; D010449:Peptide Mapping / Q000379:methods; D017434:Protein Structure, Tertiary; D011516:Prothrombin / Q000737:chemistry* / Q000378:metabolism; D013379:Substrate Specificity; D013925:Thromboplastin / Q000737:chemistry* / Q000378:metabolism
D000577:Amides; D000991:Antithrombins; D065427:Factor Xa Inhibitors; D010446:Peptide Fragments; D015943:Factor Va; D011516:Prothrombin; D013925:Thromboplastin; D015951:Factor Xa; D008239:Lysine; D000409:Alanine