RESUMEN
Human tissue factor pathway inhibitor (TFPI) is a modular protein comprised of three Kunitz type domains flanked by peptide segments that are less structured. The sequential order of the elements are: an N-terminal acidic region followed by the first Kunitz domain (K1), a linker region, a second Kunitz domain (K2), a second linker region, the third Kunitz domain (K3), and the C-terminal basic region. The K1 domain inhibits factor VIIa complexed to tissue factor (TF) while the K2 domain inhibits factor Xa. No direct protease inhibiting functions have been demonstrated for the K3 domain. Importantly, the Xa-TFPI complex is a much more potent inhibitor of the VIIa-TF than TFPI by itself. Furthermore, the C-terminal basic region of TFPI is required for rapid physiologic inhibition of coagulation and is needed for the inhibition of smooth muscle cell proliferation. Although a number of additional targets for attachment have been reported, the C-terminal basic region appears to play an important role in binding of TFPI to cell surfaces. A primary site of TFPI synthesis is endothelium and the endothelium-bound TFPI contributes to the antithrombotic potential of the vascular endothelium. Further, increased levels of plasma TFPI under septic conditions may represent endothelial dysfunction. We have proposed that the extravascular cells that synthesize TF also synthesize TFPI providing dual components necessary for the regulation of clotting in their microenvironment. Like the TF synthesis in these cells is augmented by serum, so is the case with the TFPI gene expression. TFPI gene knock out mice reveal embryonic lethality suggesting a possible role of this protein in early development. Since TF-induced coagulation is thought to play a significant role in many disease states, including disseminated intravascular clotting, sepsis, acute lung injury and cancer, recombinant TFPI may be a beneficial therapeutic agent in these disease states to attenuate pathologic clotting. The purpose of this review is to outline recent developments in the field related to the structural specificity and biology of TFPI.
Asunto(s)
Lipoproteínas , Enfermedad Aguda , Secuencia de Aminoácidos , Aminoácidos/química , Síndrome Antifosfolípido/sangre , Coagulación Sanguínea/fisiología , Enfermedades Cardiovasculares/sangre , Endotelio Vascular/metabolismo , Humanos , Lipoproteínas/química , Lipoproteínas/genética , Lipoproteínas/fisiología , Lipoproteínas/uso terapéutico , Enfermedades Pulmonares/sangre , Modelos Biológicos , Modelos Moleculares , Datos de Secuencia Molecular , Metástasis de la Neoplasia , Neoplasias/sangre , Conformación Proteica , Estructura Terciaria de Proteína , Sepsis/sangre , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Relación Estructura-Actividad , Trombofilia/sangre , Trombofilia/tratamiento farmacológico , Tromboplastina/fisiologíaRESUMEN
Human stromelysin-1 is a member of the matrix metalloproteinase (MMP) family of enzymes. The active site glutamic acid of the MMPs is conserved throughout the family and plays a pivotal role in the catalytic mechanism. The structural and functional consequences of a glutamate to glutamine substitution in the active site of stromelysin-1 were investigated in this study. In contrast to the wild-type enzyme, the glutamine-substituted mutant was not active in a zymogram assay where gelatin was the substrate, was not activated by organomercurials and showed no activity against a peptide substrate. The glutamine-substituted mutant did, however, bind to TIMP-1, the tissue inhibitor of metalloproteinases, after cleavage of the propeptide with trypsin. A second construct containing the glutamine substitution but lacking the propeptide was also inactive in the proteolysis assays and capable of TIMP-1 binding. X-ray structures of the wild-type and mutant proteins complexed with the propeptide-based inhibitor Ro-26-2812 were solved and in both structures the inhibitor binds in an orientation the reverse of that of the propeptide in the pro-form of the enzyme. The inhibitor makes no specific interactions with the active site glutamate and a comparison of the wild-type and mutant structures revealed no major structural changes resulting from the glutamate to glutamine substitution.
Asunto(s)
Sustitución de Aminoácidos/genética , Metaloproteinasa 3 de la Matriz/química , Metaloproteinasa 3 de la Matriz/genética , Modelos Moleculares , Compuestos de Aminobifenilo/farmacología , Sitios de Unión/genética , Cristalografía por Rayos X , Electroforesis en Gel de Poliacrilamida , Activación Enzimática/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Gelatina/química , Células HeLa , Humanos , Metaloproteinasa 3 de la Matriz/biosíntesis , Inhibidores de la Metaloproteinasa de la Matriz , Mutagénesis Sitio-Dirigida , Unión Proteica/fisiología , Estructura Terciaria de Proteína , Relación Estructura-Actividad , Inhibidor Tisular de Metaloproteinasa-1/químicaRESUMEN
Short chain L-3-hydroxyacyl CoA dehydrogenase (SCHAD) is a soluble dimeric enzyme critical for oxidative metabolism of fatty acids. Its primary sequence has been reported to be conserved across numerous tissues and species with the notable exception of the pig heart homologue. Preliminary efforts to solve the crystal structure of the dimeric pig heart SCHAD suggested the unprecedented occurrence of three enzyme subunits within the asymmetric unit, a phenomenon that was thought to have hampered refinement of the initial chain tracing. The recently solved crystal coordinates of human heart SCHAD facilitated a molecular replacement solution to the pig heart SCHAD data. Refinement of the model, in conjunction with the nucleotide sequence for pig heart SCHAD determined in this paper, has demonstrated that the previously published pig heart SCHAD sequence was incorrect. Presented here are the corrected amino acid sequence and the high resolution crystal structure determined for pig heart SCHAD complexed with its NAD+ cofactor (2.8 A; R(cryst) = 22.4%, R(free) = 28.8%). In addition, the peculiar phenomenon of a dimeric enzyme crystallizing with three subunits contained in the asymmetric unit is described.
Asunto(s)
3-Hidroxiacil-CoA Deshidrogenasas/química , Miocardio/enzimología , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Cristalografía por Rayos X , Cartilla de ADN , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Músculo Liso/enzimología , Conformación Proteica , Homología de Secuencia de Aminoácido , PorcinosRESUMEN
The glutamic acid-specific protease from Streptomyces griseus (SGPE) is an 18.4-kDa serine protease with a distinct preference for Glu in the P1 position. Other enzymes characterized by a strong preference for negatively charged residues in the P1 position, e.g., interleukin-1 beta converting enzyme (ICE), use Arg or Lys residues as counterions within the S1 binding site. However, in SGPE, this function is contributed by a His residue (His 213) and two Ser residues (Ser 192 and S216). It is demonstrated that proSGPE is activated autocatalytically and dependent on the presence of a Glu residue in the -1 position. Based on this observation, the importance of the individual S1 residues is evaluated considering that enzymes unable to recognize a Glu in the P1 position will not be activated. Among the residues constituting the S1 binding site, it is demonstrated that His 213 and Ser 192 are essential for recognition of Glu in the P1 position, whereas Ser 216 is less important for catalysis out has an influence on stabilization of the ground state. From the three-dimensional structure, it appears that His 213 is linked to two other His residues (His 199 and His 228), forming a His triad extending from the S1 binding site to the back of the enzyme. This hypothesis has been tested by substitution of His 199 and His 228 with other amino acid residues. The catalytic parameters obtained with the mutant enzymes, as well as the pH dependence, do not support this theory; rather, it appears that His 199 is responsible for orienting His 213 and that His 228 has no function associated with the recognition of Glu in P1.
Asunto(s)
Endopeptidasas/metabolismo , Streptomyces griseus/enzimología , Bacillus subtilis/genética , Sitios de Unión , Catálisis , Endopeptidasas/química , Endopeptidasas/genética , Genes Sintéticos , Concentración de Iones de Hidrógeno , Conformación Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMEN
Factor VIIa, in the presence of Ca2+ and tissue factor (TF), initiates the extrinsic pathway of blood coagulation. The light chain (amino acids 1-152) of factor VIIa consists of an N-terminal gamma-carboxyglutamic acid (Gla) domain followed by two epidermal growth factor-like domains, whereas the heavy chain (amino acids 153-406) contains the serine protease domain. In this study, both recombinant factor VIIa (rVIIa) and factor VIIa lacking the Gla domain were found to contain two high-affinity (Kd approximately 150 microM) Ca2+ binding sites. The rVIIa also contained approximately 6-7 low-affinity (Kd approximately 1 mM) Ca(2+)-binding sites. By analogy to other serine proteases, one of the two high affinity Ca(2+)-binding sites in factor VIIa may be formed involving Glu-210 and Glu-220 of the protease domain. In support of this, a synthetic peptide composed of residues 206-242 of factor VIIa bound one Ca2+ with Kd approximately 230 microM; however, Ca2+ binding was observed only in Tris buffer (pH 7.5) containing 1 M NaCl and not in buffer containing 0.1 M NaCl. In both low or high salt +/- Ca2+, the peptide existed as a monomer as determined by sedimentation equilibrium measurements and had no detectable secondary structure as determined by CD measurements. This indicates that subtle changes undetectable by CD may occur in the conformation of the peptide that favor calcium binding in high salt. In the presence of recombinant TF and 5 mM Ca2+, the peptide inhibited the amidolytic activity of rVIIa toward the synthetic substrate, S-2288. The concentration of the peptide required for half-maximal inhibition was approximately 5-fold higher in the low salt buffer than that in the high salt buffer. From direct binding and competitive inhibition assays of active site-blocked 125I-rVIIa binding to TF, the Kd for peptide-TF interaction was calculated to be approximately 15 microM in the high salt and approximately 55 microM in the low salt buffer containing 5 mM Ca2+. Moreover, as inferred from S-2288 hydrolysis, the Kd for VIIa.TF interaction was approximately 1.5 microM in the absence of Ca2+, and, as inferred from factor X activation studies, it was approximately 10 pM in the presence of Ca2+. Thus, Ca2+ decreases the functional Kd of VIIa.TF interaction approximately 150,000-fold.(ABSTRACT TRUNCATED AT 400 WORDS)
Asunto(s)
Calcio/metabolismo , Factor VIIa/metabolismo , Serina Endopeptidasas/metabolismo , Tromboplastina/metabolismo , Secuencia de Aminoácidos , Animales , Sitios de Unión , Bovinos , Humanos , Datos de Secuencia Molecular , Fragmentos de Péptidos/metabolismo , Fragmentos de Péptidos/farmacología , Serina Endopeptidasas/químicaRESUMEN
Factor IX consists of a gamma-carboxyglutamic acid-rich domain followed by two epidermal growth factor (EGF)-like domains and the C-terminal protease domain. To delineate the function of EGF1 domain in factor IX, we constructed three mutants: an EGF1 domain-deleted mutant (IX delta EGF1), a point mutant (IXQ50P) with a Gln-50-->Pro change, and a replacement mutant (IXPCEGF1) in which the EGF1 domain of factor IX was replaced by that of protein C. These mutants and wild-type (WT) factor IX (IXWT) were expressed in 293 kidney cells by using pRc/CMV vector. The purified proteins had the same gamma-carboxyglutamic acid content as the normal plasma factor IX (IXNP) and were activated normally by factor XIa-Ca2+. In contrast, IX delta EGF1 could not be activated by factor VIIa-tissue factor-Ca2+, and the activation of IXPCEGF1 in this system was markedly slow; however, IXQ50P was activated at a normal rate. In additional studies, both IXWT and IX delta EGF1 were rapidly converted to their respective IX alpha forms by factor Xa-phospholipid-Ca2+. Since this reaction has an absolute requirement for phospholipid, it indicates that the mutants under study are not impaired in their interactions with phospholipid. Relative coagulant activities of factor XIa-activated proteins were IXNP, 100%; IXWT, 75-85%; IX delta EGF1, < or = 1%; IXPCEGF1, < or = 2%; and IXQ50P, 6-10%. We conclude that the EGF1 domain of factor IX is required for its activation by factor VIIa-tissue factor and that the Gln-50 residue is not critical for this activation. Further, the EGF1 domain of factor IX is not essential for phospholipid binding and for its activation by factor XIa. In addition, the low coagulant activities of the activated mutants indicate that the EGF1 domain is also important in factor X activation by factor IXa-factor VIIIa-Ca(2+)-phospholipid complex.
Asunto(s)
Factor IX/metabolismo , Factor VIIa/metabolismo , Factor XIa/metabolismo , Tromboplastina/metabolismo , Ácido 1-Carboxiglutámico/análisis , Calcio/metabolismo , Activación Enzimática , Factor de Crecimiento Epidérmico/química , Factor IX/análisis , Humanos , Estructura Terciaria de ProteínaRESUMEN
Proteases specific for cleavage after acidic residues have been implicated in several disease states, including epidermolysis, inflammation, and viral processing. A serine protease with specificity toward glutamic acid substrates (Glu-SGP) has been crystallized in the presence of a tetrapeptide ligand and its structure determined and refined to an R-factor of 17% at 2.0-A resolution. This structure provides an initial description of the design of proteolytic specificity for negatively charged residues. While the overall fold of Glu-SGP closely resembles that observed in the pancreatic-type serine proteases, stabilization of the negatively charged substrate when bound to this protein appears to involve a more extensive part of the protease than previously observed. The substrate carboxylate is bound to a histidine side chain, His213, which provides the primary electrostatic compensation of the negative charge on the substrate, and to two serine hydroxyls, Ser192 and Ser216. Glu-SGP displays maximum activity at pH 8.3, and assuming normal pKa's, the glutamate side chain and His213 will be negatively charged and neutral, respectively, at this pH. In order for His213 to carry a positive charge at the optimal pH, its pKa will have to be raised by at least two units. An alternative mechanism for substrate charge compensation is suggested that involves a novel histidine triad, His213, His199, and His228, not observed in any other serine protease. The C-terminal alpha-helix, ubiquitous to all pancreatic-type proteases, is directly linked to this histidine triad and may also play a role in substrate stabilization.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Histidina/metabolismo , Serina Endopeptidasas/metabolismo , Secuencia de Aminoácidos , Sitios de Unión , Cristalografía por Rayos X , Glutamatos/metabolismo , Ácido Glutámico , Modelos Moleculares , Datos de Secuencia Molecular , Estructura Terciaria de Proteína , Homología de Secuencia de Aminoácido , Streptomyces griseus/enzimología , Especificidad por SustratoRESUMEN
Heparin-binding protein (HBP; also known as CAP37 or azurocidin) is a member of the serine proteinase family. Evolution, however, has reverted this protein into a non-proteolytic form by mutation of two of the three residues of the active-site triad. Although proteolytically inactive, the human heparin-binding protein (hHBP) is still capable of binding bovine pancreatic trypsin inhibitor (BPTI). This was demonstrated by affinity chromatography to BPTI immobilized on a solid matrix and by studies on plasmin inhibition kinetics. hHBP competes with plasmin for BPTI and this effect on plasmin inhibition has been analyzed in terms of a kinetic model. A dissociation constant, Kd = 0.1 microM, was found for the interaction between BPTI and hHBP. The hHBP provides an example of a serine proteinase which has lost its catalytic function by reverting residues of the active center while still preserving its capability of specific interactions with Kunitz inhibitors. pHBP, the porcine counterpart to hHBP, on the other hand, was incapable of BPTI binding. The structural basis for the BPTI binding to the human protein and the species difference is discussed in terms of putative three-dimensional structures of the proteins derived by comparative molecular modelling methods.
Asunto(s)
Aprotinina/metabolismo , Proteínas Sanguíneas/metabolismo , Proteínas Portadoras , Serina Endopeptidasas/metabolismo , Secuencia de Aminoácidos , Animales , Péptidos Catiónicos Antimicrobianos , Proteínas Sanguíneas/química , Bovinos , Fibrinolisina/antagonistas & inhibidores , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Alineación de Secuencia , Serina Endopeptidasas/químicaRESUMEN
A binary complex of malate dehydrogenase from the thermophilic bacterium Thermus flavus (tMDH) with NADH has been crystallized from poly(ethylene glycol) 3500, pH 8.5, yielding diffraction-quality crystals in space group P2(1)2(1)2(1). The structure was solved at 1.9-A resolution using molecular replacement and refined to an R factor of 15.8% with good geometry. The primary sequence of tMDH is 55% identical to that of cytoplasmic malate dehydrogenase (cMDH) [Birktoft, J. J., Rhodes, G., & Banaszak, L. J. (1989) Biochemistry 28, 6065-6081], and overall their three-dimensional structures are very similar. Like cMDH, tMDH crystallized as a dimer with one coenzyme bound per subunit. The coenzyme binds in the extended conformation, and most of the interactions with enzyme are similar to those in cMDH. In tMDH, small local conformational changes are caused by the replacement of a glutamic acid for the aspartic acid involved in hydrogen bonding to the adenine ribose of NADH. Comparison of tMDH with cMDH reveals that both tMDH subunits more closely resemble the B subunit of cMDH which therefore is the more likely representative of the solution conformation. While cMDH is inactivated at temperatures above about 50 degrees C, tMDH is fully active at 90 degrees C. On the basis of the X-ray crystal structure, a number of factors have been identified which are likely to contribute to the relative thermostability of tMDH compared to cMDH. The most striking of the differences involves the introduction of four ion pairs per monomer. All of these ion pairs are solvent-accessible. Three of these ion pairs are located in the dimer interface, Glu27-Lys31, Glu57-Lys168, and Glu57-Arg229, and one ion pair, Glu275-Arg149, is at the domain interface within each subunit. Additionally, we observe incorporation of additional alanines into alpha-helices of tMDH and, in one instance, incorporation of an aspartate that functions as a counterchange to an alpha-helix dipole. The possible contributions of these and other factors to protein thermostability in tMDH are discussed.
Asunto(s)
Malato Deshidrogenasa/química , Estructura Secundaria de Proteína , Thermus/enzimología , Secuencia de Aminoácidos , Estabilidad de Enzimas , Calor , Enlace de Hidrógeno , Sustancias Macromoleculares , Modelos Moleculares , Datos de Secuencia Molecular , Conformación Proteica , Homología de Secuencia de Aminoácido , Termodinámica , Difracción de Rayos XRESUMEN
On the basis of the crystal structure of the NAD-dependent cytoplasmic malate dehydrogenase (MDH) and its alignment with NADP-dependent counterparts, the loop region between beta-strand B and alpha-helix C in the dinucleotide-binding fold was predicted as a principal determinant for the coenzyme specificity. Two mutants, EX7 and EX3, of NAD-dependent MDH from Thermus flavus were constructed. In the EX7 mutant, the seven loop amino acids in positions 41-47, Glu-Ile-Pro-Gln-Ala-Met-Lys, were replaced by the corresponding loop residues in the NADP-dependent MDH from chloroplasts, Gly-Ser-Glu-Arg-Ser-Phe-Gln. In the EX3 mutant, Glu-41, Ile-42, and Ala-45 were substituted with the corresponding 3 amino acids in the NADP-dependent chloroplast MDH. In both mutations the coenzyme specificity was altered from NAD to NADP. Especially, the EX7 mutation resulted in a more than 1000-fold improvement in overall catalytic efficiency with NADPH and a 600-fold decrease in the efficiency with NADH as cofactors. Consequently, EX7 mutant was 132 times more efficient with NADPH than NADH without a large decrease in turnover number.
Asunto(s)
Coenzimas/metabolismo , Malato Deshidrogenasa/metabolismo , Mutagénesis Sitio-Dirigida , Thermus/enzimología , Secuencia de Aminoácidos , Secuencia de Bases , Cinética , Malato Deshidrogenasa/antagonistas & inhibidores , Malato Deshidrogenasa/genética , Modelos Moleculares , Datos de Secuencia Molecular , NAD/metabolismo , Oligonucleótidos , Especificidad por SustratoAsunto(s)
Factor IX/genética , Factor IX/metabolismo , Factor IXa/genética , Factor IXa/metabolismo , Secuencia de Aminoácidos , Animales , Autorradiografía/métodos , Coagulación Sanguínea , Línea Celular , Cromatografía de Afinidad/métodos , Cromatografía por Intercambio Iónico/métodos , Electroforesis en Gel de Poliacrilamida/métodos , Exones , Factor IX/aislamiento & purificación , Factor IXa/aislamiento & purificación , Humanos , Inmunoelectroforesis/métodos , Indicadores y Reactivos , Recién Nacido , Intrones , Radioisótopos de Yodo , Cinética , Modelos Moleculares , Datos de Secuencia Molecular , Conformación Proteica , Señales de Clasificación de Proteína/genética , Señales de Clasificación de Proteína/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Homología de Secuencia de Aminoácido , Serina Endopeptidasas/química , Transfección , TritioRESUMEN
Previous studies have identified a putative calcium binding site involving two glutamic acid residues located in the protease domain of coagulation factor IX. Amino acid sequence homology considerations suggest that factor VII (FVII) possesses a similar site involving glutamic acid residues 210 and 220. In the present study, we have constructed site-specific mutants of human factor VII in which Glu-220 has been replaced with either a lysine (E220K FVII) or an alanine (E220A FVII). These mutants were indistinguishable from wild-type factor VII by SDS-PAGE but only possessed 0.1% the coagulant activity of factor VII. Incubation of E220K/E220A FVII with factor Xa resulted in a slower than normal activation rate which eventually yielded a two-chain factor VIIa molecule possessing a coagulant activity of approximately 10% that of wild-type rFVIIa. Amidolytic activity measurements indicated that E220K/E220A FVIIa, unlike wild-type factor VIIa, possessed no measurable amidolytic activity toward the chromogenic substrate S-2288, even at high CaCl2 concentrations. Addition of tissue factor apoprotein, however, induced the amidolytic activity of the mutant molecule to a level 30% of that observed for wild-type factor VIIa. This tissue factor dependent enhancement of E220K/E220A FVIIa amidolytic activity was calcium dependent and required a CaCl2 concentration in excess of 5 mM for maximal rate enhancement. This was in sharp contrast to wild-type factor VIIa which required CaCl2 levels of 0.5 mM for maximal enhancement of tissue factor dependent amidolytic activity. Competition binding experiments suggest that the decrease in amidolytic and coagulant activity observed in the factor VII mutants is a direct result of impaired tissue factor binding.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Calcio/metabolismo , Endopeptidasas/metabolismo , Factor VII/metabolismo , Tromboplastina/metabolismo , Amidas/metabolismo , Secuencia de Aminoácidos , Animales , Sitios de Unión , Coagulación Sanguínea , Calcio/farmacología , Línea Celular , Cricetinae , Factor VII/química , Factor VIIa/metabolismo , Humanos , Riñón , Datos de Secuencia Molecular , Mutagénesis , Proteínas Recombinantes/metabolismo , Espectrometría de Fluorescencia , TerbioRESUMEN
Tissue plasminogen activator (t-PA) is an exceptional serine protease, because unlike most other serine protease zymogens single-chain tissue plasminogen activator (sct-PA) possesses a substantial amount of proteolytic activity. The unusual reaction of sct-PA afforded the opportunity to directly compare the active site environment of sct-PA and two-chain tissue plasminogen activator (tct-PA) in solution through the application of a series of nitroxide spin labels and fluorophores. These labels, which have been previously shown to covalently label the catalytic serine of other serine proteases, inactivated both sct-PA and tct-PA. The labels can be divided into two classes: those which form tetrahedral complexes (sulfonates) and those which form trigonal complexes (anthranilates). Those which formed tetrahedral complexes were found to be insensitive to structural differences between sct-PA and tct-PA at the active site. In contrast, those which formed trigonal complexes could differentiate and monitor the sct-PA to tct-PA conversion by fluorescence spectroscopy. Models of the structure of sct-PA and tct-PA were constructed on the basis of the known X-ray structures of other serine protease zymogen and active enzyme forms. One of the nitroxide spin labels was modeled into the sct-PA and tct-PA structures in two possible orientations, both of which could be sensitive to structural differences between sct-PA and tct-PA. These models formed the structural rationale used to explain the results obtained with the "tetrahedral" and "trigonal" probes, as well as to offer a possible explanation for the unique reactivity of sct-PA.
Asunto(s)
Activador de Tejido Plasminógeno/metabolismo , Secuencia de Aminoácidos , Espectroscopía de Resonancia por Spin del Electrón , Hidrólisis , Cinética , Modelos Moleculares , Datos de Secuencia Molecular , Conformación Proteica , Espectrometría de Fluorescencia , Marcadores de SpinRESUMEN
Factor IX is a multidomain protein and is the proenzyme of a serine protease, factor IXa, essential for hemostasis. In this report, we describe the molecular basis of hemophilia B (deficiency of factor IX activity) in five patients who have neither deletions nor rearrangements of the factor IX gene. By enzymatic amplification and sequencing of all exons and promoter regions, the following causative mutation in the protease domain of factor IX was identified in each patient: IXSchmallenberg: nucleotide 31,215G----T, Ser365Ile; IXVarel: nucleotide 31,214A----G, Ser365Gly; IXMechtal: nucleotide 31,211G----C, Asp364His; IXDreihacken: nucleotide 30,864G----A, Arg248Gln; and IXMonschau: nucleotide 30,855A----T, Glu245Val. In IXVarel, nucleotide 31,213T was also replaced by C, which results in a silent mutation (GAT----GAC) at Asp-364. Thus, this patient has a double base-pair substitution of TA to CG at nucleotides 31,213 and 31,214 but only a single amino acid change of Ser-365 to Gly. This patient also developed an antibody to factor IX during replacement therapy, which suggests that deletion of the factor IX gene is not necessary for development of the antibody in hemophilia B patients. The levels of plasma factor IX antigen in the patients ranged from 40% to 100% except for IXDreihacken (Arg248Gln), in which case it was approximately 4% of normal. The Ser365Gly and Ser365Ile mutants are nonfunctional because of lack of the active site serine residue. Mutant Asp364His is inactive because it cannot form the hydrogen bond between the carboxylate group of Asp-364 and the alpha-amino group of Val-181 generated after activation. As observed in other homologous serine proteases, this hydrogen bond is essential for maintaining the correct active site conformation in normal factor IXa (IXaN). Purified Arg248Gln had approximately 41% and Glu245Val had approximately 17% of the activity of normal factor IX (IXN) in a partial thromboplastin time (aPTT) assay. In immunodot blot experiments, the isolated Glu245Val mutant did and the Arg248Gln mutant did not bind to an anti-IXN monoclonal antibody that has been shown previously to inhibit the interaction of factor VIIIa with factor IXaN. We have recently shown that a high-affinity calcium binding site exists in the protease domain of IXN; among the proposed Ca(2+)-binding ligands is the carboxyl group of Glu-245. Further, a part of the epitope for the above antibody was shown to be contained in the 231 to 265 residue segment of factor IX.(ABSTRACT TRUNCATED AT 400 WORDS)
Asunto(s)
Factor IX/genética , Hemofilia B/genética , Mutación , Serina Endopeptidasas/química , Adulto , Arginina , Sitios de Unión , Calcio/metabolismo , Exones , Factor IX/metabolismo , Glutamatos , Ácido Glutámico , Glutamina , Humanos , Enlace de Hidrógeno , Persona de Mediana Edad , Reacción en Cadena de la Polimerasa , Regiones Promotoras Genéticas , Conformación Proteica , ValinaRESUMEN
The Fab fragment of a monoclonal antibody (mAb) reactive to the N-terminal half (residues 180-310) of the protease domain of human factor IX has been previously shown to inhibit the binding of factor IXa to its cofactor, factor VIIIa. These data suggested that this segment of factor IXa may participate in binding to factor VIIIa. We now report that the binding rate (kon) of the mAb is 3-fold higher in the presence of Ca2+ than in its absence for both factors IX and IXa; the half-maximal effect was observed at approximately 300 microM Ca2+. Furthermore, the off rate (koff) of the mAb is 10-fold higher for factor IXa than for factor IX with or without Ca2+. Moreover, like the kon for mAb binding, the incorporation of dansyl-Glu-Gly-Arg chloromethyl ketone (dEGR-CK) into factor IXa was approximately 3 times faster in the presence of Ca2+ than in its absence. Since steric factors govern the kon and the strength of noncovalent interactions governs the koff, the data indicate that the region of factor IX at residues 180-310 undergoes two separate conformational changes before expression of its biologic activity: one upon Ca2+ binding and the other upon zymogen activation. Furthermore, the dEGF-CK incorporation data suggest that both conformational changes also affect the active site residues. Analyses of the known three-dimensional structures of serine proteases indicate that in human factor IX a high-affinity Ca(2+)-binding site may be formed by the carboxyl groups of glutamates 235 and 245 and by the main chain carbonyl oxygens of residues 237 and 240. In support of this conclusion, a synthetic peptide including residues 231-265 was shown to bind Ca2+ with a Kd of approximately 500 microM. This peptide also bound to the mAb, although with approximately 500-fold reduced affinity. Moreover, like factor IX, the peptide bound to the mAb more strongly (approximately 3-fold) in the presence of Ca2+ than in its absence. Thus, it appears that a part of the epitope for the mAb described above is contained in the proposed Ca(2+)-binding site in the protease domain of human factor IX. This proposed site is analogous to the Ca(2+)-binding site in trypsin and elastase, and it may be involved in binding factor IXa to factor VIIIa.
Asunto(s)
Factor IX/fisiología , Secuencia de Aminoácidos , Anticuerpos Monoclonales/inmunología , Afinidad de Anticuerpos , Sitios de Unión , Calcio/metabolismo , Activación Enzimática , Precursores Enzimáticos/metabolismo , Factor IX/ultraestructura , Humanos , Técnicas Inmunológicas , Técnicas In Vitro , Modelos Moleculares , Datos de Secuencia Molecular , Movimiento (Física) , Péptidos/metabolismo , Conformación ProteicaRESUMEN
Malate dehydrogenase from mutant strain F428 of the thermophilic bacterium Thermus flavus has now been crystallized from polyethylene glycol 8000 in a form suitable for diffraction studies. The protein crystallizes in the orthorhombic P2(1)2(1)2(1) space group with unit cell dimensions a = 71.8 A, b = 88.6 A, c = 119.0 A. The asymmetric unit consists of one homodimer of molecular mass 67,000 Da. The X-ray diffraction extends beyond 1.7 A and a full data set to 1.9 A has been collected.
Asunto(s)
Malato Deshidrogenasa/genética , Mutagénesis , Thermus/genética , Cristalización , Malato Deshidrogenasa/química , Conformación Proteica , Thermus/enzimología , Difracción de Rayos XRESUMEN
Thrombin is a serine protease that acts as a procoagulant by clotting fibrinogen and activating platelets and as an anticoagulant by activating protein C in a thrombomodulin-dependent reaction. Fibrinogen and thrombomodulin bind competitively to an anion-binding exosite on thrombin. We prepared recombinant normal human thrombin and mutant thrombins with single amino acid substitutions in order to localize and distinguish the fibrinogen- and thrombomodulin-binding sites. Normal and mutant thrombins had similar amidolytic activity. Thrombin K52E had approximately 2.5-fold increased protein C-activating activity but only approximately 17% of normal fibrinogen-clotting activity. Thrombin R70E had normal fibrinogen-clotting activity but only approximately 7% of normal protein C-activating activity. Thrombin R68E had markedly reduced activity in both assays. Decreased activation of protein C correlated with decreased binding affinity for thrombomodulin, and ability to activate platelets correlated directly with fibrinogen-clotting activity. These results demonstrate that thrombins with predominantly anticoagulant or procoagulant activity can be created by mutagenesis and that thrombomodulin- and fibrinogen-binding sites on thrombin may overlap but are not identical.
Asunto(s)
Fibrinógeno/metabolismo , Receptores de Superficie Celular/metabolismo , Trombina/metabolismo , Secuencia de Aminoácidos , Animales , Línea Celular , Simulación por Computador , Humanos , Cinética , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Agregación Plaquetaria/efectos de los fármacos , Conformación Proteica , Protrombina/genética , Protrombina/metabolismo , Receptores de Trombina , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacología , Trombina/genética , Trombina/farmacología , TransfecciónRESUMEN
The synthesis of nitroxide spin-labeled derivatives of S-acetoacetyl-CoA, S-acetoacetylpantetheine, and S-acetoacetylcysteamine is described. These compounds are active substrates of L-3-hydroxyacyl-CoA dehydrogenase [(S)-3-hydroxyacyl-CoA:NAD+ oxidoreductase, EC 1.1.1.35] exhibiting vmax values from 20% to 70% of S-acetoacetyl-CoA itself. S-Acetoacetylpantetheine and S-acetoacetylcysteamine form binary complexes with the enzyme and exhibit ESR spectra typical for immobilized nitroxides. In the case of spin-labeled pantetheine, the radical is more mobile. When spin-labeled substrates are bound simultaneously to each active site of this dimeric enzyme, spin-spin interactions differentiate between two alternate orientations of the substrate [Birktoft, J.J., Holden, H.M., Hamlin, R., Xuong, N.H., & Banaszak, L.J. (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 8262-8266]. The fatty acid moiety is thought to be located in a cleft between two domains whereas a large part of the CoA moiety probably extends into the solution. NAD+, spin-labeled at N6 of the adenine ring, is an active coenzyme of L-3-hydroxyacyl-CoA dehydrogenase (60% vmax). Complexes with the enzyme exhibit ESR spectra typical of highly immobilized nitroxides. Binding of coenzyme NAD+ causes conformational changes of the binary enzyme/substrate complex as revealed by changes in the ESR spectrum of spin-labeled S-acetoacetylpantetheine.