Factor XIa: Difference between revisions
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Thrombin-catalyzed proteolysis of factor XI involves crucial interations with Glu-66, Lys-83 and Gln-84 of the A1 domain (this ensures maximum proximity to the <scene name='Sandbox/Activation_loop/1'>activation loop</scene> of factor XI) of the factor XI molecule through its exosites I and II regions <ref>PMID:16699514</ref>. Thus binding of thrombin to one subunit of the zymogen dimer promotes cleavage of the bond between <scene name='Sandbox/Arg369-ile370/1'> Arg369-Ile370</scene> contained in the <scene name='Sandbox/Activation_loop/1'>activation loop</scene> of factor XI. The <scene name='Sandbox/Activation_loop/1'>activation loop</scene> (residues 370-376) consequently undergoes the greatest conformational change as Ile-370 is displaced ~20Å from its position in factor XI and inserts into the activation pocket of factor XIa producing the oxyanion hole in the active site of the protease <ref>PMID:14523451</ref>. | Thrombin-catalyzed proteolysis of factor XI involves crucial interations with Glu-66, Lys-83 and Gln-84 of the A1 domain (this ensures maximum proximity to the <scene name='Sandbox/Activation_loop/1'>activation loop</scene> of factor XI) of the factor XI molecule through its exosites I and II regions <ref>PMID:16699514</ref>. Thus binding of thrombin to one subunit of the zymogen dimer promotes cleavage of the bond between <scene name='Sandbox/Arg369-ile370/1'> Arg369-Ile370</scene> contained in the <scene name='Sandbox/Activation_loop/1'>activation loop</scene> of factor XI. The <scene name='Sandbox/Activation_loop/1'>activation loop</scene> (residues 370-376) consequently undergoes the greatest conformational change as Ile-370 is displaced ~20Å from its position in factor XI and inserts into the activation pocket of factor XIa producing the oxyanion hole in the active site of the protease <ref>PMID:14523451</ref>. | ||
==Substrate Recognition and Cleavage== | ===Substrate Recognition and Cleavage=== | ||
The primary substrate of factor XIa is another zymogen, factor IX which is cleavage sequentially at the peptides bonds between Arg145-Ala146 and Arg180-Val181 of factor IX to release an activation peptide <ref>PMID:17676929</ref>. Recognition of the substrate (factor IX) involves residues different from the <scene name='Sandbox/Active_site/2'>active site</scene> residues. In the inactive zymogen (factor XI), the highly conserved <scene name='Sandbox/Arg_184/1'>Arg-184</scene> is buried in the interface between the apple domains and the catalytic domain where it interacts with <scene name='Sandbox/3_residues/1'>three residues</scene>: Ser-268 from the A3 domain and Asp-488 and Asn-566 in the catalytic domain. Thus following activation, <scene name='Sandbox/Arg_184/1'>Arg-184</scene> is believed to constitute a switch which undegoes a conformational change breaking its interaction with Ser-268, Asp-488 and Asn 566 facilitating the protease interaction with factor IX <ref>PMID:16699514</ref>. | The primary substrate of factor XIa is another zymogen, factor IX which is cleavage sequentially at the peptides bonds between Arg145-Ala146 and Arg180-Val181 of factor IX to release an activation peptide <ref>PMID:17676929</ref>. Recognition of the substrate (factor IX) involves residues different from the <scene name='Sandbox/Active_site/2'>active site</scene> residues. In the inactive zymogen (factor XI), the highly conserved <scene name='Sandbox/Arg_184/1'>Arg-184</scene> is buried in the interface between the apple domains and the catalytic domain where it interacts with <scene name='Sandbox/3_residues/1'>three residues</scene>: Ser-268 from the A3 domain and Asp-488 and Asn-566 in the catalytic domain. Thus following activation, <scene name='Sandbox/Arg_184/1'>Arg-184</scene> is believed to constitute a switch which undegoes a conformational change breaking its interaction with Ser-268, Asp-488 and Asn 566 facilitating the protease interaction with factor IX <ref>PMID:16699514</ref>. | ||
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Similar to other members of the serine protease family, factor XIa bears the two β-barrels topology unique to chymotrypsin-like proteases which are linked through a central loop. Thus cleavage at <scene name='Sandbox/Arg369-ile370/1'> Arg369-Ile370</scene> of the zymogen generates a functional enzyme. the [http://en.wikipedia.org/wiki/Catalytic_triad catalytic triad] residues Ser-557, Asp-462 and His-413 constitute the <scene name='Sandbox/Active_site/2'>active site</scene> of factor XIa. | Similar to other members of the serine protease family, factor XIa bears the two β-barrels topology unique to chymotrypsin-like proteases which are linked through a central loop. Thus cleavage at <scene name='Sandbox/Arg369-ile370/1'> Arg369-Ile370</scene> of the zymogen generates a functional enzyme. the [http://en.wikipedia.org/wiki/Catalytic_triad catalytic triad] residues Ser-557, Asp-462 and His-413 constitute the <scene name='Sandbox/Active_site/2'>active site</scene> of factor XIa. | ||
===The General Base Mechanism of Serine Proteases=== | ===The Conventional General Base Mechanism of Serine Proteases=== | ||
The catalytic triad residues assume their correct orientation following the proteolysis of the activation peptide to form a competent enzyme. The carbonyl group of the scissile bond to be broken is positioned closely to the β-OH group of Ser195. His57 is also located in close proximity to abstract a proton from β-OH group of Ser195. H-bonding interaction between the carboxyl group of Asp102 and Nδ1-H of His57 allows His57 to act as a general base. | The catalytic triad residues assume their correct orientation following the proteolysis of the activation peptide to form a competent enzyme. The carbonyl group of the scissile bond to be broken is positioned closely to the β-OH group of Ser195. His57 is also located in close proximity to abstract a proton from β-OH group of Ser195. H-bonding interaction between the carboxyl group of Asp102 and Nδ1-H of His57 allows His57 to act as a general base. | ||
Thus, the Nε2 of His57 acts a the general base to enhance the nucleophilicity of the catalytic Ser195-OH by abstracting its proton. The acylation phase occurs with the attack of the carbonyl of the scissile bond generation the transition state intermediate. Also known as the tetrahedral intermediate, the negative oxygen ion in this structure is thought to be stabilized by the oxyanion hole formed by the amide backbone hydrogens of Ser195 and Gly193. The collapse of this intermediate involves the abstraction of the Nε2-H of His57 (general acid) by the leaving group P1' nitrogen of the substrate forming the first product and an acylenzyme intermediate<ref>PMID:332063</ref>. | Thus, the Nε2 of His57 acts a the general base to enhance the nucleophilicity of the catalytic Ser195-OH by abstracting its proton. The acylation phase occurs with the attack of the carbonyl of the scissile bond generation the transition state intermediate. Also known as the tetrahedral intermediate, the negative oxygen ion in this structure is thought to be stabilized by the oxyanion hole formed by the amide backbone hydrogens of Ser195 and Gly193. The collapse of this intermediate involves the abstraction of the Nε2-H of His57 (general acid) by the leaving group P1' nitrogen of the substrate forming the first product and an acylenzyme intermediate<ref>PMID:332063</ref>. | ||
During the deacylation phase of the mechanism,water molecule is activated to act as the nucleophile by the abstration of a proton by the Nε2 of His57. The resulting nucleophilic OH anion attacks the carbonyl of the acylenzyme to generate another tetrahedral intermediate whose subsequent collapse releases the carboxylic half of the second product<ref>PMID:4372018</ref>.[[Image:ChargeRelay.png| thumb|650px| The Charge Relay Mechanism of serine proteases]] | During the deacylation phase of the mechanism,water molecule is activated to act as the nucleophile by the abstration of a proton by the Nε2 of His57. The resulting nucleophilic OH anion attacks the carbonyl of the acylenzyme to generate another tetrahedral intermediate whose subsequent collapse releases the carboxylic half of the second product<ref>PMID:4372018</ref>.[[Image:ChargeRelay.png| thumb|650px| The Charge Relay Mechanism of serine proteases]] | ||
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In a model not widely accepted by some enzymologists, Pery and colleagues proposed that in the LBHB-facilitated general base mechanism, Nε2 of His57 abstracts a proton from the β-OH of Ser195 in the transition state for its subsequent addition to the substrate-carbonyl group. Thus, the imadazole-carboxylic acid interaction is stablilzed during the formation of the tetrahedral intermediate by the <scene name='Factor_XIa/Lbhb/1'>LBHB</scene> formed by the interaction between Nδ1 of His57 and β-COOH of Asp102<ref>PMID:7661899</ref>. Proponents of this theory suggest that, the catalytic properties of α-chymotrypsin-mediated by LBHB would be lost when His57 is modified. As expected, N-methylation of His57 led to 0.2 million-fold reduction in acylation of α-chymotrypsin<ref>PMID:5126468</ref>. Single substitution mutation (Asp102->Asn) in trypsin resulted in a 1/10th decrease in catalytic activity in the mutant<ref>PMID:3303334</ref>. | In a model not widely accepted by some enzymologists, Pery and colleagues proposed that in the LBHB-facilitated general base mechanism, Nε2 of His57 abstracts a proton from the β-OH of Ser195 in the transition state for its subsequent addition to the substrate-carbonyl group. Thus, the imadazole-carboxylic acid interaction is stablilzed during the formation of the tetrahedral intermediate by the <scene name='Factor_XIa/Lbhb/1'>LBHB</scene> formed by the interaction between Nδ1 of His57 and β-COOH of Asp102<ref>PMID:7661899</ref>. Proponents of this theory suggest that, the catalytic properties of α-chymotrypsin-mediated by LBHB would be lost when His57 is modified. As expected, N-methylation of His57 led to 0.2 million-fold reduction in acylation of α-chymotrypsin<ref>PMID:5126468</ref>. Single substitution mutation (Asp102->Asn) in trypsin resulted in a 1/10th decrease in catalytic activity in the mutant<ref>PMID:3303334</ref>. | ||
===The His Flip Mechanism of Serine Proteases=== | |||
[[Image:His flip modified.gif|right|thumb|650px| '''The His Flip mechanism of Serine proteases''']] | |||
Studies on the central role of His57 and its orientation as regards functioning as the general base and general acid, led to the proposal of the '''His Moving''' mechanism. Earlier kinetics studies using subtilisin as the model organism revealed that, whereas the kcat for ester hydrolysis in 50% N,N-dimethylformamide (DMF) remains unchanged, hydrolysis of amides was 2-fold lower. | |||
To verify this phenomenon, Kidd and colleagues<ref>PMID:10048334</ref> examined the crystal structure of subtilisin in 50% DMF. Diffraction data revealed that, the imidazole ring of His64, had rotated ~180 degrees around the Cβ-Cγ bond hence His64 did not interact with the other active site residues ( Ser221 and Asp32. NMR studies confirmed that the unique resonance of His57 due to the low barrier hydrogen bond between the His64 and Asp32 is absent in 50% DMF. Using fast magic-angle spinning solid-state NMR spectroscopy, Bachovachin in 2001<ref> PMID:3542033 </ref>,proposed the presence of Ser214-His57 H-bond and the resonance of His57 as evidence supporting the His Moving mechanim. | |||
According to this mechanism. the Nε1-H is within H-bonding distance of the γO of Ser195 following the formation of the tetrahedral intermediate. Rotation of His57 orients Nε1-H the closer to the leaving group NH. New H-bonding partners are formed in place to the disrupted H-bonding between Nδ1-H & Asp102 and Cε1 & Ser214<ref>PMID:10048334</ref><ref> PMID:3542033 </ref>. | |||
Opponents of the His flip mechanism argue that, the disruption and reforming of several H-bonds in the short-life of the transition state is least favorable and the restricted movement of His57 by the P2 and P1’ residues makes this mechanism unlikely<ref> PMID:2271520</ref>. The mechanism is also contrary to the theory of least motion which proposes, that enzyme reactions proceed with an economy of movement<ref> PMID:11170405</ref>. | |||
==Evolutionary conservation== | ==Evolutionary conservation== | ||