Role of the residues of the 39-loop in determining the substrate and inhibitor specificity of factor IXa.
The activation of antithrombin (AT) by heparin facilitates the exosite-dependent interaction of the serpin with factors IXa (FIXa) and Xa (FXa), thereby improving the rate of reactions by 300- to 500-fold. Relative to FXa, AT inhibits FIXa with approximately 40-fold slower rate constant. Structural data suggest that differences in the residues of the 39-loop (residues 31-41) may partly be responsible for the differential reactivity of the two proteases with AT. This loop is highly acidic in FXa, containing three Glu residues at positions 36, 37, and 39. By contrast, the loop is shorter by one residue in FIXa (residue 37 is missing), and it contains a Lys and an Asp at positions 36 and 39, respectively. To determine whether differences in the residues of this loop contribute to the slower reactivity of FIXa with AT, we prepared an FIXa/FXa chimera in which the 39-loop of the protease was replaced with the corresponding loop of FXa. The chimeric mutant cleaved a FIXa-specific chromogenic substrate with normal catalytic efficiency, however, the mutant exhibited approximately 5-fold enhanced reactivity with AT specifically in the absence of the cofactor, heparin. Further studies revealed that the FIXa mutant activates factor X with approximately 4-fold decreased k(cat) and approximately 2-fold decreased K(m), although the mutant interacted normally with factor VIIIa. Based on these results we conclude that residues of the 39-loop regulate the cofactor-independent interaction of FIXa with its physiological inhibitor AT and substrate factor X.
Yang
Likui
L
Edward A. Doisy Department of Biochemistry and Molecular Biology, St. Louis University School of Medicine, St. Louis, Missouri 63104, USA.
Manithody
Chandrashekhara
C
Qureshi
Shabir H
SH
Rezaie
Alireza R
AR
D000991:Antithrombins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D002460:Cell Line; D015949:Factor IXa / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D015951:Factor Xa / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D011485:Protein Binding / Q000235:genetics; D017433:Protein Structure, Secondary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity / Q000235:genetics
D000991:Antithrombins; D011993:Recombinant Fusion Proteins; D015949:Factor IXa; D015951:Factor Xa