Factor Xa

From Proteopedia

Revision as of 15:46, 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 epidermal 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 triad 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]

Enzyme Mechanism

General Serine Protease Mechanism

During the acylation half of the reaction Ser195 attacks the carbonyl of the peptide bond to be broken within the substrate, His57 assists by acting as a general base, deprotonating Ser195, to yield a tetrahedral intermediate. This intermediate is stabilized through hydrogen bonding interaction with the backbone amides in the oxyanion hole (Gly193 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 C-terminal portion of the substrate. The deacylation portion repeats the same sequence. A water molecule deprotonated by His57 attacks the acyl enzyme, to yield another tetrahedral intermediate, prior to yielding the N-terminal portion of the protein.^[4]

Controversial Mechanisms

His Flip Mechanisms

As stated previously, His57 removes a proton from Ser195 and transfers it to the leaving group. It can be argued that the protonated His57 could reprotonate Ser195 and regenerate the substrate. One hypothesis is that protonated His57 flips such that N1 proton could easily protonate the leaving group. ^[5] This flipped conformation has been observed in another group of serine proteases, subtilisin, in a 50% dimethylformamide solution. ^[6] However, there are 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. ^[7]These observations disfavor the His flip mechanism.

Other observations have suggested that Ser195 must move 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.

Low Barrier Hydrogen Bonds

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 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. ^{[8] [9]} This would enhance catalysis in the first step of the reaction. However, other groups argue against the role of an LBHB in serine protease catalysis. They hypothesize that electrostatic and van der Waals interactions in the active site are responsible for stabilizing the transition state. ^{[10] [11] [12]}

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 WW. 15N NMR spectroscopy of hydrogen-bonding interactions in the active site of serine proteases: evidence for a moving histidine mechanism. Biochemistry. 1986 Nov 18;25(23):7751-9. PMID:3542033
6. ↑ 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.
7. ↑ Brady K, Wei AZ, Ringe D, Abeles RH. Structure of chymotrypsin-trifluoromethyl ketone inhibitor complexes: comparison of slowly and rapidly equilibrating inhibitors. Biochemistry. 1990 Aug 21;29(33):7600-7. PMID:2271520
8. ↑ Frey PA, Whitt SA, Tobin JB. A low-barrier hydrogen bond in the catalytic triad of serine proteases. Science. 1994 Jun 24;264(5167):1927-30. PMID:7661899
9. ↑ Frey, Perry A. Strong hydrogen bonding in chymotrypsin and other serine proteases. Journal of Physical Organic Chemistry (2004), 17(6-7), 511-520.
10. ↑ Warshel A. Electrostatic origin of the catalytic power of enzymes and the role of preorganized active sites. J Biol Chem. 1998 Oct 16;273(42):27035-8. PMID:9765214
11. ↑ Kollman PA, Kuhn B, Donini O, Perakyla M, Stanton R, Bakowies D. Elucidating the nature of enzyme catalysis utilizing a new twist on an old methodology: quantum mechanical-free energy calculations on chemical reactions in enzymes and in aqueous solution. Acc Chem Res. 2001 Jan;34(1):72-9. PMID:11170358
12. ↑ Ash EL, Sudmeier JL, De Fabo EC, Bachovchin WW. A low-barrier hydrogen bond in the catalytic triad of serine proteases? Theory versus experiment. Science. 1997 Nov 7;278(5340):1128-32. PMID:9353195