The conformational switch from the factor X zymogen to protease state mediates exosite expression and prothrombinase assembly.
Zymogens of the chymotrypsin-like serine protease family are converted to the protease state following insertion of a newly formed, highly conserved N terminus. This transition is accompanied by active site formation and ordering of several surface loops in the catalytic domain. Here we show that disruption of this transition in factor X through mutagenesis (FXa(I16L) and FXa(V17A)) not only alters active site function, but also significantly impairs Na(+) and factor Va binding. Active site binding was improved in the presence of high NaCl or with saturating amounts of factor Va membranes, suggesting that allosteric linkage exists between these sites. In line with this, irreversible stabilization of FXa(I16L) with Glu-Gly-Arg-chloromethyl ketone fully rescued FVa binding. Furthermore, the K(m) for prothrombin conversion with the factor Xa variants assembled into prothrombinase was unaltered, whereas the k(cat) was modestly reduced (3- to 4-fold). These findings show that intramolecular activation of factor X following the zymogen to protease transition not only drives catalytic site activation but also contributes to the formation of the Na(+) and factor Va binding sites. This structural plasticity of the catalytic domain plays a key role in the regulation of exosite expression and prothrombinase assembly.
Toso
Raffaella
R
Department of Pediatrics, Division of Hematology, The Children's Hospital of Philadelphia, Pennsylvania 19104, USA.
Zhu
Hua
H
Camire
Rodney M
RM
D001665:Binding Sites / Q000502:physiology; D004789:Enzyme Activation / Q000502:physiology; D004792:Enzyme Precursors / Q000235:genetics / Q000378:metabolism*; D005165:Factor V / Q000235:genetics / Q000378:metabolism*; D015943:Factor Va / Q000235:genetics / Q000378:metabolism*; D015951:Factor Xa / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D016296:Mutagenesis; D011485:Protein Binding / Q000502:physiology; D020836:Protein Structure, Quaternary / Q000502:physiology; D017434:Protein Structure, Tertiary / Q000502:physiology; D012964:Sodium / Q000378:metabolism; D046249:Transfer RNA Aminoacylation
D004792:Enzyme Precursors; C022475:prothrombinase complex; D015943:Factor Va; D005165:Factor V; D012964:Sodium; D015951:Factor Xa