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1.
J Biol Chem ; 288(47): 33611-33619, 2013 Nov 22.
Artigo em Inglês | MEDLINE | ID: mdl-24068708

RESUMO

Allosteric conformational changes in antithrombin induced by binding a specific heparin pentasaccharide result in very large increases in the rates of inhibition of factors IXa and Xa but not of thrombin. These are accompanied by CD, fluorescence, and NMR spectroscopic changes. X-ray structures show that heparin binding results in extension of helix D in the region 131-136 with coincident, and possibly coupled, expulsion of the hinge of the reactive center loop. To examine the importance of helix D extension, we have introduced strong helix-promoting mutations in the 131-136 region of antithrombin (YRKAQK to LEEAAE). The resulting variant has endogenous fluorescence indistinguishable from WT antithrombin yet, in the absence of heparin, shows massive enhancements in rates of inhibition of factors IXa and Xa (114- and 110-fold, respectively), but not of thrombin, together with changes in near- and far-UV CD and (1)H NMR spectra. Heparin binding gives only ∼3-4-fold further rate enhancement but increases tryptophan fluorescence by ∼23% without major additional CD or NMR changes. Variants with subsets of these mutations show intermediate activation in the absence of heparin, again with basal fluorescence similar to WT and large increases upon heparin binding. These findings suggest that in WT antithrombin there are two major complementary sources of conformational activation of antithrombin, probably involving altered contacts of side chains of Tyr-131 and Ala-134 with core hydrophobic residues, whereas the reactive center loop hinge expulsion plays only a minor additional role.


Assuntos
Antitrombina III/química , Fator IXa/química , Fator Xa/química , Mutação , Regulação Alostérica/genética , Antitrombina III/genética , Antitrombina III/metabolismo , Dicroísmo Circular , Fator IXa/genética , Fator IXa/metabolismo , Fator Xa/genética , Fator Xa/metabolismo , Humanos , Ressonância Magnética Nuclear Biomolecular , Estrutura Secundária de Proteína
2.
Biochemistry ; 49(13): 2918-24, 2010 Apr 06.
Artigo em Inglês | MEDLINE | ID: mdl-20218626

RESUMO

The formation of a blood clot involves the interplay of thrombin, fibrinogen, and Factor XIII. Thrombin cleaves fibrinopeptides A and B from the N-termini of the fibrinogen Aalpha and Bbeta chains. Fibrin monomers are generated that then polymerize into a noncovalently associated network. By hydrolyzing the Factor XIII activation peptide segment at the R37-G38 peptide bond, thrombin assists in activating the transglutaminase FXIIIa that incorporates cross-links into the fibrin clot. In this work, the kinetic effects of introducing fibrinogen Aalpha character into the FXIII AP segment were examined. Approximately 25% of fibrinogen Aalpha is phosphorylated at Ser3, producing a segment with improved binding to thrombin. FXIII AP ((22)AEDDL(26)) has sequence properties in common with Fbg Aalpha ((1)ADSpGE(5)). Kinetic benefits to FXIII AP cleavage were explored by extending FXIII AP (28-41) to FXIII AP (22-41) and examining peptides with D24, D24S, D24Sp, and D24Sp P27G. These modifications did not provide the same kinetic advantages that were observed with Fbg Aalpha (1-20) S3p. Such results further emphasize that FXIII AP derives most of its substrate specificity from the P(9)-P(1) segment. To enhance the kinetic properties of FXIII AP (28-41), we introduced substitutions at the P(9), P(4), and P(3) positions. Studies reveal that FXIII AP (28-41) V29F, V34G, V35G exhibits kinetic improvements that are comparable to those of FXIII AP V29F, V34L and approach those of Fbg Aalpha (7-20). Selective changes to the FXIII AP segment sequence may be used to design FXIII species that can be activated more or less readily.


Assuntos
Fator XIII/metabolismo , Fibrinogênio/metabolismo , Ativação Enzimática , Fator XIII/genética , Humanos , Hidrólise , Cinética , Mutagênese Sítio-Dirigida , Fragmentos de Peptídeos , Especificidade por Substrato , Trombina/metabolismo , Transglutaminases/metabolismo
3.
Biochemistry ; 46(9): 2444-52, 2007 Mar 06.
Artigo em Inglês | MEDLINE | ID: mdl-17286389

RESUMO

In the last stages of coagulation, thrombin helps to activate Factor XIII. The resultant transglutaminase introduces covalent cross-links into fibrin thus promoting clot stability. To better understand the roles of individual thrombin residues in recognition and hydrolysis of the Factor XIII activation peptide, mutations within thrombin's aryl and apolar binding site were explored. The thrombin mutants W215A, E217A, W215A/E217A, L99A, and I174A were examined through HPLC kinetics against the substrates FXIII (28-41) V34 AP and FXIII (28-41) V34L AP. Several mutants responded differently to FXIII (28-41) V34 AP vs the cardioprotective V34L AP. W215 provides an important platform for binding and directing FXIII APs for proper hydrolysis. Loss of this platform leads to decreases in kinetics, particularly to the kcat of FXIII V34L AP. E217 also plays a supporting role, but the E217A mutation is not as detrimental as W215A. W215A/E217A is unfavorable for both activation peptides and its coupling effect has been characterized. This mutant can readily bind the peptides but cannot orient them for effective hydrolysis. Kinetic studies with I174A indicate that this thrombin residue is more crucial for interactions with the larger V34L AP segment. The L99A mutation causes deleterious effects to binding and hydrolysis of both APs. The V34L, however, is able to partially compensate for the loss perhaps by increasing contact within the aryl and apolar sites. Understanding how specific FXIII and thrombin residues participate in binding and control hydrolysis may lead to the design of coagulation enzymes whose degree of activation and optimal target site can be controlled.


Assuntos
Fator XIII/metabolismo , Trombina/metabolismo , Sequência de Aminoácidos , Cromatografia Líquida de Alta Pressão , Fator XIII/química , Fator XIII/genética , Humanos , Hidrólise , Cinética , Dados de Sequência Molecular , Mutagênese , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz
4.
Arch Biochem Biophys ; 445(1): 36-45, 2006 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-16364233

RESUMO

Thrombin cleaves fibrinopeptides A and B from fibrinogen leading to the formation of a fibrin network that is later covalently crosslinked by Factor XIII (FXIII). Thrombin helps activate FXIII by catalyzing hydrolysis of the FXIII activation peptides (AP). In the current work, the role of exosites in the ternary thrombin-FXIII-fibrin(ogen) complex was further explored. Hydrolysis studies indicate that thrombin predominantly utilizes its active site region to bind extended Factor XIII AP (FXIII AP 33-64 and 28-56) leaving the anion-binding exosites for fibrin(ogen) binding. The presence of fibrin-I leads to improvements in the K(m) for hydrolysis of FXIII AP (28-41), whereas peptides based on the cardioprotective FXIII V34L sequence exhibit less reliance on this cofactor. Surface plasmon resonance measurements reveal that d-Phe-Pro-Arg-chloromethylketone-thrombin binds to fibrinogen faster than to FXIII a(2) and dissociates from fibrinogen more slowly than from FXIII a(2). This system of thrombin exosite interactions with differing affinities promotes efficient clot formation.


Assuntos
Fator XIII/química , Fibrina/química , Fibrinogênio/química , Trombina/química , Clorometilcetonas de Aminoácidos/química , Sequência de Aminoácidos , Sítios de Ligação , Cromatografia Líquida de Alta Pressão , Humanos , Hidrólise , Dados de Sequência Molecular , Fragmentos de Peptídeos/química , Ligação Proteica , Especificidade por Substrato , Ressonância de Plasmônio de Superfície
5.
Biochemistry ; 43(14): 4150-9, 2004 Apr 13.
Artigo em Inglês | MEDLINE | ID: mdl-15065858

RESUMO

In blood coagulation, thrombin helps to activate factor XIII by cleaving the activation peptide at the R37-G38 peptide bond. The residues N-terminal to the scissile bond are important in determining rates of hydrolysis. Solution studies of wild-type and mutant peptides of factor XIII AP (28-37) suggest residues P(4)-P(1) are most critical in substrate recognition. By contrast, the X-ray crystal structure of FXIII AP (28-37) displays all of the residues, P(10)-P(1), interacting with the thrombin active site in a conformation similar to that of fibrinogen Aalpha (7-16) [Sadasivan, C., and Yee, V. C. (2000) J. Biol. Chem. 275, 36942-36948]. Peptides were therefore synthesized with the N-terminal P(10)-P(6) residues removed to further characterize interactions of thrombin with factor XIII activation peptides. The truncations have no adverse effects on thrombin's ability to bind and to hydrolyze the shortened peptides. The wild-type FXIII AP (33-41) V34 sequence actually exhibits a decrease in K(m) relative to the longer (28-41) sequence whereas the cardioprotective FXIII AP (33-41) V34L exhibits a further increase in k(cat) relative to its longer parent sequence. One-dimensional proton line broadening NMR and 2D transferred-NOESY studies indicate that the shortened peptides maintain similar bound conformations as their FXIII AP (28-37) counterparts. Furthermore, the distinctive NOE between the L34 and P36 side chains is preserved. Kinetic and NMR studies thus reveal that the N-terminal portions of FXIII AP (28-37) (V34 and V34L) are not necessary for effective interaction with the thrombin active site surface. FXIII activation peptides bind to thrombin in a manner more like PAR1 than fibrinogen Aalpha.


Assuntos
Fator XIII/metabolismo , Fragmentos de Peptídeos/metabolismo , Peptídeos/metabolismo , Deleção de Sequência , Trombina/metabolismo , Substituição de Aminoácidos/genética , Sítios de Ligação/genética , Fator XIII/química , Humanos , Hidrólise , Peptídeos e Proteínas de Sinalização Intercelular , Cinética , Leucina/genética , Ressonância Magnética Nuclear Biomolecular , Fragmentos de Peptídeos/síntese química , Fragmentos de Peptídeos/genética , Peptídeos/síntese química , Peptídeos/genética , Conformação Proteica , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , Especificidade por Substrato , Valina/genética
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