Factor Xa

From Proteopedia

Revision as of 00:23, 26 April 2010

Introduction

Factor X is a vitamin K-dependent glycoprotein that is synthesized in the liver. 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 factor VIIa-tissue factor (extrinsic Xase complex) and factor IXa-factor VIIIa (intrinsic Xase complex).^[1]

Factor Xa, along with factor Va, calcium, and a phospholipid membrane surface form the prothrombinase complex, to cleave prothrombin to its active form, thrombin.^[1]

Structure

The factor Xa light chain contains a γ-carboxyglutamic acid (Gla) domain (11 gla residues) as well as two epidermial growth factor (EGF)-like domains.^[2] The Gla domain is mediates calcium dependent binding of factor X to negatively charged phopholipid membrane surfaces. Recent crystal structures suggest that the N-terminal epidermal growth factor (EGF)-like domain is flexibly, while the second EGF domain maintains contacts with the catalytic domain. ^[2]

The factor Xa heavy chain contains the activation peptide and trypsin-like serine protease domain. ^[3]

Catalytic Triad

The catalytic consists of His57, Ser195, and Asp102 located near each other, and each playing an important role. The serine donates an OH group to act as a nucleophile and attack the carbonyl group of the peptide bond that will be broken within the substrate. Histidine coordinates the attack of the peptide bond by accepting the hydrogen from the serine –OH group with a pair of electrons on nitrogen. Aspartic acid contains a carboxyl group to hydrogen bond with the histidine to make the pair of electrons more electronegative.

Substrate Recognition Sites

The S1 pocket determines binding selectivity for factor Xa. It is formed by loops in residues 214-220 and 189-195 that are linked by a Cys220-Cys191 disulfide bond. Residues 225-228 form the lower portion of the pocket.

The S2 site of factor Xa is formed by the 90s loop which is positioned adjacent to His57. Consistent with glycine as the P2 element in prothrombin, S2 is a small, shallow pocket.

S4 pocket is formed between the 90s and 170s loops and bind an Ile. This region contains 3 ligand binding domains. The hydrophobic box is located at the entrance to S4 and contains Phe174, Tyr99 and Trp215, which form a deep aryl-binding pocket. The cationic hole is formed by Glu97 and the backbone carbonyl of Lys96. The water site is composed of the hydrophobic side chains of Thr98, Ile175 and Thr177 and traps a water molecule. ^[3]

Activation domain

Enzyme Mechanism

General Serine Protease Mechanism

During the acylation portion of the reaction Ser195 attacks the carbonyl of the peptide substrate, His57 assists by acting as a general base to yield a tetrahedral intermediate. Asp102 stabilizes the His57-H+ through hydrogen bonding. The tetrahedral intermediate oxyanion is stabilized by interacting with amine groups of the peptide backbone. Upon collapse of the tetrahedral the amine leaving group is expelled with the asstance of His57-H+ acting as a general acid to yield the acylenzyme intermediate. The deacylation portion repeats the same sequence. Water is assisted by His57 to attack the acyl enzyme, to yield another tetrahedral intermediate. Upon intermediate collapse Ser195 and the carboxylic acid product are expelled. ^[4]

Low Barrier Hydrogen Bonds

Moving His Mechanisms

As stated previously, His57 removes a proton from Ser195 and transfers it to the leaving group. It can be argued that His57-H+ could reprotonate Ser195 and regenerate the substrate. One solution is that His57-H+ flips such that N1 proton could easily protonate the leaving group. This flipped conformation has been observed in another group of serine proteases, subtilisin, in a 50% dimethylformamide solution. ^[5] There are also several arguments against the His flip mechanism. Flipping of His57 would require breaking and reforming many hydrogen bonds while the short lived tetrahedral intermediate is present. Also, His57 is sterically hindered by the P2 and P1’ residues of the peptide substrates. With these consideration the His flip mechanism seems to fail in three dimensions.

Other observations have suggested that Ser195 moves at least 1Å in order to form the tetrahedral intermediate. This conformation change would cause Ser195 and His57 to be oriented away from each other upon formation of the tetrahedral intermediate, preventing reprotonation.

The Hydrolytic Water

Stabilization of the Tetrahedral Intermediate

Kinetics

Inactivation

Related Enzymes

References

1. ↑ ^{1.0 1.1} Greer, John (2008). Wintrobe's Clinical Hematology, p. 545-546. Lippincott Williams & Wilkins. ISBN 0781765072.
2. ↑ ^{2.0 2.1} Padmanabhan K, Padmanabhan KP, Tulinsky A, Park CH, Bode W, Huber R, Blankenship DT, Cardin AD, Kisiel W. Structure of human des(1-45) factor Xa at 2.2 A resolution. J Mol Biol. 1993 Aug 5;232(3):947-66. PMID:8355279 doi:http://dx.doi.org/10.1006/jmbi.1993.1441
3. ↑ ^{3.0 3.1} Rai R, Sprengeler PA, Elrod KC, Young WB. Perspectives on factor Xa inhibition. Curr Med Chem. 2001 Feb;8(2):101-19. PMID:11172669
4. ↑ Hedstrom L. Serine protease mechanism and specificity. Chem Rev. 2002 Dec;102(12):4501-24. PMID:12475199
5. ↑ Bachovchin, W. Contributions of NMR spectroscopy to the study of hydrogen bonds in serine protease active sites. Magnetic Resonance in Chemistry; (2001); 39(Spec. Issue); 199-213.