Factor VIIa: Difference between revisions
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The binding epitope of TF to FVIIa is a stripe running along the whole length of the TF protein. | The binding epitope of TF to FVIIa is a stripe running along the whole length of the TF protein. | ||
The Gla domain of FVIIa binds to the C-domain of TF. The interaction is mainly hydrophobic termed the “hydrophobic stack”. EGF1 binds a single Ca++ ion and packs into a groove formed by the two modules of TF. This interface is the largest and contributes the most energetically in binding of the cofactor to the protease domain13. EGF2 and the catalytic domain interact with the N-domain of TF. Two “lock and key” interactions are observed. One is the side chain of Phe50 of TF which is trapped in the pocket formed by the end of EGF2 domain of FVIIa. Second, the side chain Met306 of FVIIa is enclosed by TF residues Arg74, Phe76, Glu92, Leu94. This Met306 residue is responsible for the thermodynamic coupling to the active site and is unique to FVIIa. Mutation of this residue results in the failure of TF to decrease the dissociation rate of the enzyme from the cofactor. Cofactor interactions, specifically through Met306 lead to subtle changes which then influence the position of Asp331 (check). Asp331 is the specificity-determining residue in the binding pocket. Constraint and stabilization promote formation of a hydrogen bond between the amide of Arg315 (170C) and carbonyl of Gly372 (223). When bound to TF the activation region of FVIIa contains a large number of hydrogen bonds between the main chain and side chain atoms. At least 11 water molecules are identified in the catalytic domain region. An interesting observation is that the center of this region contains water, in contrast to the hydrophobic interactions in the other two interface regions. These hydrophilic interactions may be more efficient at mediating TF affinity and substantial conformational changes to induce activity of FVIIa. Therefore TF and cations are obligatory cofactors in the allosteric regulation of FVIIa activity by stabilizing the disordered, felxible FVIIa and restraining the enzyme for catalytic activity. | The Gla domain of FVIIa binds to the C-domain of TF. The interaction is mainly hydrophobic termed the “hydrophobic stack”. EGF1 binds a single Ca++ ion and packs into a groove formed by the two modules of TF. This interface is the largest and contributes the most energetically in binding of the cofactor to the protease domain13. EGF2 and the catalytic domain interact with the N-domain of TF. Two “lock and key” interactions are observed. One is the side chain of Phe50 of TF which is trapped in the pocket formed by the end of EGF2 domain of FVIIa. Second, the side chain Met306 of FVIIa is enclosed by TF residues Arg74, Phe76, Glu92, Leu94. This Met306 residue is responsible for the thermodynamic coupling to the active site and is unique to FVIIa. Mutation of this residue results in the failure of TF to decrease the dissociation rate of the enzyme from the cofactor. Cofactor interactions, specifically through Met306 lead to subtle changes which then influence the position of Asp331 (check). Asp331 is the specificity-determining residue in the binding pocket. Constraint and stabilization promote formation of a hydrogen bond between the amide of Arg315 (170C) and carbonyl of Gly372 (223). When bound to TF the activation region of FVIIa contains a large number of hydrogen bonds between the main chain and side chain atoms. At least 11 water molecules are identified in the catalytic domain region. An interesting observation is that the center of this region contains water, in contrast to the hydrophobic interactions in the other two interface regions. These hydrophilic interactions may be more efficient at mediating TF affinity and substantial conformational changes to induce activity of FVIIa. Therefore TF and cations are obligatory cofactors in the allosteric regulation of FVIIa activity by stabilizing the disordered, felxible FVIIa and restraining the enzyme for catalytic activity. | ||
===Catalytic domain=== | |||
There are three steps serine proteases take to hydrolyze an amide bond: 1)activation of amide bonds by the interaction of the general acid with the carbonyl oxygen of the substrtate amide bond which disrupts resonance stabilization 2) activation of water by general base 3)activation of amines by protonation before expulsion. Serine proteases hydrolyze amide bonds with rates of 1010 –fold higher than the uncatalyzed reactions. | |||
In FVIIa Ser344(195) of the catalytic triad is activated by a His193(57), or the general base, which itself is stabilized by a hydrogen bond to Asp242(102). These reactions result in a formation of a tetrahedral intermediate and the oxyanion hole. The oxyanion hole is stabilized by interactions with main chain NHs. The activated Ser344 then attacks the scissile bond of the substrate. The general base His193 transfers the abstracterd proton from Ser to the amine leaving group, the tetrahedral intermediate (transition state) collapses and an acylenzyme intermediate is formed releasing the product. The general base His193 abstracts a proton from water as it attacks the acylenzyme to again form a tetrahedral intermediate. His193 then acts as an acid and protonates Ser344 releasing the product acid and regenerates the enzyme. This reaction is largely possible by having a His193 with a pKa ~7 necessary for deprotonation15, a hydrogen bonding network or “the charge relay system” activating Ser344 for nucleophilic attack, stabilization of the negatively charged oxyanion of the tetrahedral intermediate by the main chain NHs of Ser344 and Gly342(193). The kinetics are described in three steps: 1) binding of enzyme to substrate (k+1, k-1), 2) acylation of enzyme (k2), and 3) deacylation (k3) INSERT FIGURE 21.10.3 | |||
