Factor Xa: Difference between revisions
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<StructureSection load='2PR3' size='350' side='right' scene='' caption='Human factor X heavy chain (grey) and light chain (green) complex with pyrrolydine derivative inhibitor and Ca+2 ions (green) (PDB code [[2pr3]])'> | <StructureSection load='2PR3' size='350' side='right' scene='' caption='Human factor X heavy chain (grey) and light chain (green) complex with pyrrolydine derivative inhibitor and Ca+2 ions (green) (PDB code [[2pr3]])'> | ||
==Introduction== | ==Introduction== | ||
[[Image:Coagulation full.svg.png|left|thumb|450px|The coagulation cascade.]] | |||
{{Clear}} | |||
'''Factor X''' is a vitamin K-dependent [http://en.wikipedia.org/wiki/Glycoprotein glycoprotein] that is synthesized in the liver. [http://en.wikipedia.org/wiki/Zymogen Zymogen] factor X circulates in plasma as a 2 chain molecule composed of a disulfide linked light chain (Mr = 16500) and heavy chain (Mr = 42,000). Factor X is activated to '''factor Xa''' by cleavage of the activation peptide. This reaction is catalyzed by [http://en.wikipedia.org/wiki/Factor_VIIa factor VIIa]-[http://en.wikipedia.org/wiki/Tissue_factor tissue factor] (extrinsic Xase complex) and [http://en.wikipedia.org/wiki/Factor_ixa factor IXa]-[http://en.wikipedia.org/wiki/Factor_viiia factor VIIIa] (intrinsic Xase complex).<ref name="Greer">Greer, John (2008). ''Wintrobe's Clinical Hematology'', p. 545-546. Lippincott Williams & Wilkins. ISBN 0781765072.</ref> | '''Factor X''' is a vitamin K-dependent [http://en.wikipedia.org/wiki/Glycoprotein glycoprotein] that is synthesized in the liver. [http://en.wikipedia.org/wiki/Zymogen Zymogen] factor X circulates in plasma as a 2 chain molecule composed of a disulfide linked light chain (Mr = 16500) and heavy chain (Mr = 42,000). Factor X is activated to '''factor Xa''' by cleavage of the activation peptide. This reaction is catalyzed by [http://en.wikipedia.org/wiki/Factor_VIIa factor VIIa]-[http://en.wikipedia.org/wiki/Tissue_factor tissue factor] (extrinsic Xase complex) and [http://en.wikipedia.org/wiki/Factor_ixa factor IXa]-[http://en.wikipedia.org/wiki/Factor_viiia factor VIIIa] (intrinsic Xase complex).<ref name="Greer">Greer, John (2008). ''Wintrobe's Clinical Hematology'', p. 545-546. Lippincott Williams & Wilkins. ISBN 0781765072.</ref> | ||
'''Factor Xa''', along with [http://en.wikipedia.org/wiki/Factor_va factor Va], calcium, and a phospholipid membrane surface to form the [http://en.wikipedia.org/wiki/Prothrombinase prothrombinase complex], and cleave [http://en.wikipedia.org/wiki/Prothrombin prothrombin] to its active form, [http://en.wikipedia.org/wiki/Prothrombin thrombin].<ref name="Greer" /> | '''Factor Xa''', along with [http://en.wikipedia.org/wiki/Factor_va factor Va], calcium, and a phospholipid membrane surface to form the [http://en.wikipedia.org/wiki/Prothrombinase prothrombinase complex], and cleave [http://en.wikipedia.org/wiki/Prothrombin prothrombin] to its active form, [http://en.wikipedia.org/wiki/Prothrombin thrombin].<ref name="Greer" /> | ||
==Relevance== | |||
Factor Xa is inhibited by [[Apixaban]] and [[Rivaroxaban]] which are anticoagulant medications. See also [[Anticoagulants]]. | |||
==Structure== | ==Structure== | ||
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<scene name='Factor_Xa/Transparent_-_no_inhib_s4/2'>S4 pocket</scene> is formed between the 90s and 170s loops and binds an Ile 12. This region contains 3 ligand binding domains. The <scene name='Factor_Xa/Transparent_-_no_inhib_phob_bo/4'>hydrophobic box</scene> is located at the entrance to S4 and contains Phe174, Tyr99 and Trp215, which form a deep aryl-binding pocket. The <scene name='Factor_Xa/Transparent_-_no_inhib_oxianio/3'>cationic hole</scene> is formed by the backbone carbonyl and side chain of Glu97 and the backbone carbonyl of Lys96. The <scene name='Factor_Xa/Transparent_-_no_inhib-_h2o_si/3'>water site</scene> is composed of the hydrophillic side chains of Thr98, Ile175 and Thr177 and traps a water molecule. <ref name="Inhib" /> | <scene name='Factor_Xa/Transparent_-_no_inhib_s4/2'>S4 pocket</scene> is formed between the 90s and 170s loops and binds an Ile 12. This region contains 3 ligand binding domains. The <scene name='Factor_Xa/Transparent_-_no_inhib_phob_bo/4'>hydrophobic box</scene> is located at the entrance to S4 and contains Phe174, Tyr99 and Trp215, which form a deep aryl-binding pocket. The <scene name='Factor_Xa/Transparent_-_no_inhib_oxianio/3'>cationic hole</scene> is formed by the backbone carbonyl and side chain of Glu97 and the backbone carbonyl of Lys96. The <scene name='Factor_Xa/Transparent_-_no_inhib-_h2o_si/3'>water site</scene> is composed of the hydrophillic side chains of Thr98, Ile175 and Thr177 and traps a water molecule. <ref name="Inhib" /> | ||
The cation-pi interaction is a strong, non-covalent bond formed by electrostatic interactions between the side chains of aromatic residues and various cations. These bonds are characterized by the fact that sp2 carbons are more electronegative then hydrogen, and 6 local Cδ- - Hδ+ bond dipoles are created around the benzene ring. Collectively, these dipoles create an accumulation of negative charge in the center of the ring and a belt of positive charge around the edge. This charge distribution allows for cation binding to the center of the ring, however, if the ring is not properly positioned, the cation will be repulsed by the positive charge of the outer ring. The S4 binding pocket of Factor Xa is formed from three aromatic residues, tyrosine 99, phenylalanine 174, tryptophan 215, sufficiently rich in properly positioned pi-electrons that it is not only a hydrophobic pocket, but also forms a cation recognition site. Many factor Xa inhibitors have a basic residue binding in this pocket, when protonated, cation-pi interactions are formed. | The cation-pi interaction is a strong, non-covalent bond formed by electrostatic interactions between the side chains of aromatic residues and various cations. These bonds are characterized by the fact that sp2 carbons are more electronegative then hydrogen, and 6 local Cδ- - Hδ+ bond dipoles are created around the benzene ring. Collectively, these dipoles create an accumulation of negative charge in the center of the ring and a belt of positive charge around the edge. This charge distribution allows for cation binding to the center of the ring, however, if the ring is not properly positioned, the cation will be repulsed by the positive charge of the outer ring. The S4 binding pocket of Factor Xa is formed from three aromatic residues, tyrosine 99, phenylalanine 174, tryptophan 215, sufficiently rich in properly positioned pi-electrons that it is not only a hydrophobic pocket, but also forms a cation recognition site. Many factor Xa inhibitors have a basic residue binding in this pocket, when protonated, cation-pi interactions are formed. | ||
*<scene name='95/953982/Cv/2'>Apixaban binding site</scene>. | |||
*<scene name='95/954035/Cv/5'>Rivaroxaban binding site</scene>. | |||
Hydrogen bonds form between the carbonyl oxygen of Ser214 and the NH of the P1 (Arg 14) residue, the NH of | Hydrogen bonds form between the carbonyl oxygen of Ser214 and the NH of the P1 (Arg 14) residue, the NH of Trp215 and the carbonyl of P3 (Asp 113) and the carbonyl of Gly216 and the NH of P3 (Asp 113). These interactions are a general feature of chymotrypsin-like proteases and are critical for efficient substrate hydrolysis. | ||
Trp215 and the carbonyl of P3 (Asp 113) and the carbonyl of Gly216 and the NH of P3 (Asp 113). These interactions are a general feature of chymotrypsin-like proteases and are critical for efficient substrate hydrolysis. | |||
The precise interactions of the P' side chains have not been defined. The P1' and P3' residues point in the same direction as a consequence of the beta sheet alignment of the substrate, so that the S1' and S3' sites overlap. The S1'/S3' sites are bounded by His57, the 60’s loop and the 40’s loop. The P2' residue points in the opposite direction and may interact with the 150’s loop. | The precise interactions of the P' side chains have not been defined. The P1' and P3' residues point in the same direction as a consequence of the beta sheet alignment of the substrate, so that the S1' and S3' sites overlap. The S1'/S3' sites are bounded by His57, the 60’s loop and the 40’s loop. The P2' residue points in the opposite direction and may interact with the 150’s loop. | ||
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===General Serine Protease Mechanism=== | ===General Serine Protease Mechanism=== | ||
During the acylation half of the reaction His57 acts as a general base to remove a proton from Ser195, allowing it to attack the carbonyl of the peptide bond to be broken within the substrate, to yield the first tetrahedral intermediate. The negative oxygen ion of the tetrahedral intermediate is stabilized through hydrogen bonding with the oxyanion hole (Gly192 and Ser195). Asp102 stabilizes the protonated His57 through hydrogen bonding. His57 protonates the amine of the scissile bond, promoting formation of the acylenzyme and release of the N-terminal portion of the substrate. | During the acylation half of the reaction His57 acts as a general base to remove a proton from Ser195, allowing it to attack the carbonyl of the peptide bond to be broken within the substrate, to yield the first tetrahedral intermediate. The negative oxygen ion of the tetrahedral intermediate is stabilized through hydrogen bonding with the oxyanion hole (Gly192 and Ser195). Asp102 stabilizes the protonated His57 through hydrogen bonding. His57 protonates the amine of the scissile bond, promoting formation of the acylenzyme and release of the N-terminal portion of the substrate. | ||
The deacylation portion repeats the same sequence. A water molecule is deprotonated by His57 and attacks the acyl enzyme, to yielding a second tetrahedral intermediate. Again, the tetrahedral intermediate is stabilized by the oxyanion hole. Upon collapse of the tetrahedral intermediate, the C-terminal portion of the protein is released.<ref name="specificity">PMID:12475199</ref>[[Image:Serine protease mechanism.gif.png| | The deacylation portion repeats the same sequence. A water molecule is deprotonated by His57 and attacks the acyl enzyme, to yielding a second tetrahedral intermediate. Again, the tetrahedral intermediate is stabilized by the oxyanion hole. Upon collapse of the tetrahedral intermediate, the C-terminal portion of the protein is released.<ref name="specificity">PMID:12475199</ref> | ||
[[Image:Serine protease mechanism.gif.png|400px|center|'''Serine protease reaction mechanism''' <ref> www.bmolchem.wisc.edu/</ref> />]] | |||
===Controversial Mechanisms=== | ===Controversial Mechanisms=== | ||
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====Low Barrier Hydrogen Bonds==== | ====Low Barrier Hydrogen Bonds==== | ||
< | <scene name='Factor_Xa/Lbhb/1'>Possible LBHB between His57 and Asp102</scene>. The mechanism by which the transition state is stabilized has been the topic of recent debate. Some groups suggest that His57 and Asp102 form and especially strong hydrogen bond, called a [http://en.wikipedia.org/wiki/Low-barrier_hydrogen_bond low barrier hydrogen bond (LBHB)]. They hypothesize that this hydrogen bond could promote formation of the transition state by stabilizing the Asp –His association and enhancing the bascisity of His57. <ref> PMID: 7661899</ref> <ref name="Frey alone">Frey, Perry A. Strong hydrogen bonding in chymotrypsin and other serine proteases. Journal of Physical Organic Chemistry (2004), 17(6-7), 511-520. </ref> This would enhance catalysis in the first step of the reaction. Formation of a LBHB requires a ΔpKa of approximately zero and a donor-to-acceptor distance of less then 2.65 Å for a nitrogen-oxygen pair like His57 and Asp102. Unlike a standard hydrogen bond, in which the hydrogen is located on the donor atom, a hydrogen in a LBHB is located equidistant between the 2 atoms. <ref name="subang"> PMID: 16834383 </ref> In 1998 Kuhn and colleagues published a crystal structure of ''Bacillus lentus'' subtilisn, another serine proetase, with 0.78 Å resolution at pH 5.9. The structure showed a distance of approximately 2.62 Å between the His57 nitrogen and the Asp102 oxygen, suggesting a LBHB. <ref> PMID: 9753430 </ref> | ||
The mechanism by which the transition state is stabilized has been the topic of recent debate. Some groups suggest that His57 and Asp102 form and especially strong hydrogen bond, called a [http://en.wikipedia.org/wiki/Low-barrier_hydrogen_bond low barrier hydrogen bond (LBHB)]. They hypothesize that this hydrogen bond could promote formation of the transition state by stabilizing the Asp –His association and enhancing the bascisity of His57. <ref> PMID: 7661899</ref> <ref name="Frey alone">Frey, Perry A. Strong hydrogen bonding in chymotrypsin and other serine proteases. Journal of Physical Organic Chemistry (2004), 17(6-7), 511-520. </ref> This would enhance catalysis in the first step of the reaction. Formation of a LBHB requires a ΔpKa of approximately zero and a donor-to-acceptor distance of less then 2.65 Å for a nitrogen-oxygen pair like His57 and Asp102. Unlike a standard hydrogen bond, in which the hydrogen is located on the donor atom, a hydrogen in a LBHB is located equidistant between the 2 atoms. <ref name="subang"> PMID: 16834383 </ref> In 1998 Kuhn and colleagues published a crystal structure of ''Bacillus lentus'' subtilisn, another serine proetase, with 0.78 Å resolution at pH 5.9. The structure showed a distance of approximately 2.62 Å between the His57 nitrogen and the Asp102 oxygen, suggesting a LBHB. <ref> PMID: 9753430 </ref> | |||
A more recent crystal structure of α-Lytic protease, published in 2006 with 0.82 Å resolution argues against both the his flip mechanism and the presence of a LBHB between His57 and Asp102 (2.755 Å in this structure). Fuhrmann ''et al'' suggests that a LBHB may have been present in the subtilisin strucutre, it is not required for the serine protease mechanism. Instead they state that the chymotrypsin-like proteases may use a network of optimized hydrogen bonds to position the stabilize the tetrahedral intermediate and position the catalytic triad. Ser195 undergoes a shift of ~1Å upon protonation of His57 that destabilizes the His57-Ser195 H-bond. This conformation change would prevent His57 from reprotonating Ser195 leading to regeneration of the substrate.<ref name="subang" /> | A more recent crystal structure of α-Lytic protease, published in 2006 with 0.82 Å resolution argues against both the his flip mechanism and the presence of a LBHB between His57 and Asp102 (2.755 Å in this structure). Fuhrmann ''et al'' suggests that a LBHB may have been present in the subtilisin strucutre, it is not required for the serine protease mechanism. Instead they state that the chymotrypsin-like proteases may use a network of optimized hydrogen bonds to position the stabilize the tetrahedral intermediate and position the catalytic triad. Ser195 undergoes a shift of ~1Å upon protonation of His57 that destabilizes the His57-Ser195 H-bond. This conformation change would prevent His57 from reprotonating Ser195 leading to regeneration of the substrate.<ref name="subang" /> | ||