Characterization of the P2‘ and P3‘ Specificities of Thrombin Using Fluorescence-Quenched Substrates and Mapping of the Subsites by Mutagenesis,

Bonniec, Bernard Le; Myles, Timothy; Johnson, Troy A.; Cg, Knight; Tapparelli, Carlo; Stone,

doi:10.1021/bi952701s

Cited by 87 publications

(87 citation statements)

References 75 publications

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“…In thrombin-catalyzed reactions, a net increase occurs in the isotopic fractionation factors for hydrogen bonds at solvation sites in the ratedetermining step. This effect is elicited by interactions at the leaving groups' binding site or substrate specificity site, which observation is well supported by other studies of P' site interactions in catalysis (32)(33)(34) by and inhibition(35) of proteases. The proton inventory technique has been particularly revealing about the importance of solvent reorganization in substrate binding and leaving group release in thrombin-catalyzed reactions.…”

supporting

confidence: 80%

Deuterium Solvent Isotope Effect and Proton-Inventory Studies of Factor Xa-Catalyzed Reactions

Zhang

Kovach

2006

Biochemistry

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Kinetic solvent isotope effects (KSIE) for the Factor Xa (FXa)-catalyzed activation of prothrombin in the presence and absence of Factor Va (FVa) and 5.0 ×10 −5 M phospholipid vesicles, are slightly inverse, 0.82−0.93, when substrate concentrations are at 0.2 K m . This is consistent with ratedetermining association of the enzyme-prothrombin assembly, rather than rate-limiting chemical transformation. FVa is known to effect a major conformational change to expose the first scissile bond in prothrombin, which is the likely event triggering a major solvent rearrangement. At prothrombin concentrations >5 K m the KSIE is 1.6 ± 0.3, when FXa is in 1 : 1 ratio with FVa, but becomes increasingly inverse, 0.30 ± 0.05 and 0.19 ± 0.04, when FXa : FVa 1:4, with increasing FXa concentration and substrate concentration. The rate-determining step changes with the conditions, but the chemical step is not limiting under any circumstance. This corroborates the proposed predominance of the meizothrombin pathway when FXa is well saturated with the prothrombin complex. In contrast, the FXa-catalyzed hydrolysis of N-α-Z-D-Arg-Gly-ArgpNA·2HCl (S-2765) and H-D-Ile-L-Pro-L-Arg-pNA.HCl (S-2288) is most consistent with twoproton bridges forming at the transition state between Ser 195 OγH and His 57 Nε2 and His 57 Nδ1 and Asp 102 COOβ-at the active site, with transition state fractionation factors of ϕ 1 = ϕ 2 = 0.57 ± 0.07 and ϕ S = 0.78 ± 0.16 for solvent rearrangement for S-2765, and ϕ 1 = ϕ 2 = 0.674 ± 0.001 for S-2288 under enzyme saturation with substrate at pH 8.40 and 25.0 ± 0.1 °C. The rate-determining step(s) in these reactions is most likely the cleavage of the C-N bond and departure of the leaving group.Factor Xa (FXa) is one of the best characterized of the serine proteases in the cardiovascular system.(1-6) It catalyzes the efficient activation of a large precursor protein, prothrombin, to form α-thrombin.(1;7;8) Although several proteolytic enzymes can bring about this reaction, it is FXa that performs this key function under physiological conditions. On the basis of extensive investigations, the prothrombin complex is known to consist of phospholipid surface, prothrombin, Ca 2+ and factor Va (FVa). (8)(9)(10)(11)(12) This construct facilitates the activation of prothrombin by ∼3 × 10 5 -fold over catalysis by FXa alone.(13;14) The activation of human prothrombin involves the hydrolysis of two peptide bonds, Arg 322 -Ile 323 followed by Arg 273 -Thr 274 . In contrast, when FXa acts alone, bond cleavage occurs in the opposite order. Different intermediates are formed in the two pathways that have also been studied independently.(9; 14) More in-depth kinetic studies lately revealed that an appreciable fraction of prothrombin is channeled directly to thrombin product without releasing intermediate products. (14)

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confidence: 80%

Deuterium Solvent Isotope Effect and Proton-Inventory Studies of Factor Xa-Catalyzed Reactions

Zhang

Kovach

2006

Biochemistry

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“…Table I shows sequences for Fibrinogen A␣ (amino acids 7-20) (Fbg A␣- (7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20)), Factor XIII activation peptide (aa 28 -41) (FXIII AP-(28 -41)), D-Phe-Pro-Arg-chloromethylketone (PPACK), and thrombin receptor (aa 32-45) (PAR1- (32)(33)(34)(35)(36)(37)(38)(39)(40)(41)(42)(43)(44)(45)). In each peptide, there is an Arg residue at the P 1 position 2 that binds within the catalytic cleft of thrombin forming a salt bridge with thrombin amino acid 3 Asp 189 .…”

mentioning

confidence: 99%

“…FXIII AP-(28 -41), PAR1-(32-45), and PPACK each contain a proline at the P 2 position. X-ray crystal studies of PAR1- (32)(33)(34)(35)(36)(37)(38)(39)(40)(41)(42)(43)(44)(45) and PPACK reveal that thrombin contains a hydrophobic surface that can accommodate this proline (9,10). Fbg A␣- (7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20), by contrast, contains a Val at the P 2 position instead of the Pro.…”

mentioning

confidence: 99%

Examining Thrombin Hydrolysis of the Factor XIII Activation Peptide Segment Leads to a Proposal for Explaining the Cardioprotective Effects Observed with the Factor XIII V34L Mutation

Trumbo

Maurer

2000

Journal of Biological Chemistry

130

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In the blood coagulation cascade, thrombin cleaves fibrinopeptides A and B from fibrinogen revealing sites for fibrin polymerization that lead to insoluble clot formation. Factor XIII stabilizes this clot by catalyzing the formation of intermolecular cross-links in the fibrin network. Thrombin activates the Factor XIII a 2 dimer by cleaving the Factor XIII activation peptide segment at the Arg 37 -Gly 38 peptide bond. Using a high performance liquid chromatography assay, the kinetic constants K m , k cat , and k cat /K m were determined for thrombin hydrolysis of fibrinogen A␣-(7-20), Factor XIII activation peptide-(28 -41), and Factor XIII activation peptide-(28 -41) with a Val 34 to Leu substitution. This Val to Leu mutation has been correlated with protection from myocardial infarction. In the absence of fibrin, the Factor XIII activation peptide-(28 -41) exhibits a 10-fold lower k cat /K m value than fibrinogen A␣-(7-20). With the Factor XIII V34L mutation, decreases in K m and increases in k cat produce a 6-fold increase in k cat /K m relative to the wild-type Factor XIII sequence. A review of the x-ray crystal structures of known substrates and inhibitors of thrombin leads to a hypothesis that the new Leu generates a peptide with more extensive interactions with the surface of thrombin. As a result, the Factor XIII V34L is proposed to be susceptible to wasteful conversion of zymogen to activated enzyme. Premature depletion may provide cardioprotective effects.Fibrinogen is composed of three chains A␣, B␤, and ␥ arranged into the dimer (A␣B␤␥) 2 . In blood coagulation, the serine protease thrombin cleaves the N-terminal portions of the A␣ and B␤ chains. For the A␣ chain, cleavage occurs at the Arg 16 -Gly 17 peptide bond and fibrinopeptide A (FpA) 1 is released; whereas, for the B␤ chain, cleavage occurs at the Arg 14 -Gly 15 peptide bond and fibrinopeptide B (FpB) is released. Removal of the fibrinopeptides leads to exposure of fibrin polymerization sites that react to form an insoluble blood clot (reviewed in Ref. 1).Activated Factor XIII helps stabilize this clot structure by catalyzing the formation of intermolecular ␥-glutamyl-⑀-lysine cross-links in the fibrin network and in fibrin-enzyme complexes. Factor XIII is a member of a family of enzymes known as transglutaminases that have a catalytic triad, similar to cysteine proteases, composed of amino acids Cys 314 , His 373, and Asp 396 . In plasma, Factor XIII is expressed as a zymogen of the form a 2 b 2 . In the presence of thrombin and calcium, the a 2 unit is released and activated. By contrast, platelet Factor XIII is expressed as the zymogen a 2 unit (reviewed in Ref. 2).The Factor XIII a 2 dimer contains in the N-terminal portion of each monomer a sequence known as the activation peptide (3, 4). Each activation peptide segment crosses the dimer interface and extends over the catalytic site of the opposing Factor XIII a subunit. Cleavage of the activation peptide segments by thrombin at the Arg 37 -Gly 38 peptide bond aids in exposure of the Fact...

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“…In the case of the co-expression of GB1KID and PKA, the serine residue in the thrombin cleavage site can be phosphorylated since it is contained within a PKA recognition site, Arg (or X)-Arg-X-Ser/Thr-X [12]. The serine residue is not necessary for the thrombin cleavage reaction as long as an acidic residue is not placed at the position of the serine residue in the thrombin cleavage site [13]. As the N-terminal residue of our KID construct is (nonacidic) histidine that originates from the NdeI restriction enzyme site, the serine residue was deleted by QuickChange site-directed mutagenesis method (Stratagene).…”

Section: Methodsmentioning

confidence: 99%

“…It is also suitable for easy and inexpensive labeling with 13 C and/or 15 N for NMR studies. Figure 1 outlines the GB1-fusion co-expression system, a derivative of pET22b (Novagen) with two cloning and ribosomal binding sites.…”

Section: Introductionmentioning

confidence: 99%

Overexpression of post-translationally modified peptides in Escherichia coli by co-expression with modifying enzymes

Sugase¹,

Landes²,

Wright³

et al. 2008

Protein Expression and Purification

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SynopsisPost-translational modification plays crucial roles in signal transduction in eukaryotic cells. To elucidate the biological function of a protein with a specific post-translational modification, it is necessary to isolate the modified protein. However, it is difficult to incorporate a modified amino acid into a specific position of a protein, in particular, in a large-scale preparation. In order to prepare post-translationally modified proteins in Escherichia coli (E. coli), we have constructed coexpression vectors that contain protein and corresponding enzyme genes. The protein and enzyme are co-expressed in the same E. coli cells and the protein is post-translationally modified in vivo. By using this system, the transcriptional activator cyclic-AMP-response-element-binding protein (CREB) was phosphorylated at Ser-133 and the hypoxia-inducible factor-1α (HIF-1α) was hydroxylated at Asn-803 in E. coli. Although the constructs of the proteins we used are very flexible and susceptible to degradation by proteases in E. coli when they are expressed alone, the B1 domain of streptococcal protein G (GB1) fused to the N-terminus of the proteins increased the yields dramatically. Site-specific phosphorylation of CREB and hydroxylation of HIF-1α were confirmed by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) and NMR. Our GB1-fusion co-expression system can be used in the same way as conventional protein expression in E. coli, making it a flexible and economical method to produce a large amount of a post-translationally modified protein. Keywordsphosphorylation; hydroxylation; co-expression; Escherichia coli; B1 domain of protein G (GB1); CREB; HIF-1α INTRODUCTIONPost-translational modification plays a crucial role in signal transduction in eukaryotic cells by introducing diversity and versatility into proteins translated from genomic DNA sequences [1]. Post-translational modification extends the range of function of the proteins by attaching other biochemical functional groups such as phosphate, hydroxyl group, acetate, lipids, or carbohydrates, causing changes in intrinsic activities of the proteins such as activation, inactivation or change in other properties such as conformational change. Despite the *To whom correspondence should be addressed (email yamout@scripps.edu). † Current address: Suntory Institute for Bioorganic Research, 1-1-1 Wakayamadai, Shimamoto-cho, Mishima-gun, Osaka 618-8503, Japan.Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. importance of post-translational modifications in biology, the production of large quantities of mod...

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Characterization of the P₂‘ and P₃‘ Specificities of Thrombin Using Fluorescence-Quenched Substrates and Mapping of the Subsites by Mutagenesis^,

Cited by 87 publications

References 75 publications

Deuterium Solvent Isotope Effect and Proton-Inventory Studies of Factor Xa-Catalyzed Reactions

Deuterium Solvent Isotope Effect and Proton-Inventory Studies of Factor Xa-Catalyzed Reactions

Examining Thrombin Hydrolysis of the Factor XIII Activation Peptide Segment Leads to a Proposal for Explaining the Cardioprotective Effects Observed with the Factor XIII V34L Mutation

Overexpression of post-translationally modified peptides in Escherichia coli by co-expression with modifying enzymes

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