Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.4.21.69 (APC)
 16,337 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Lys60f has been proposed to limit the S1' substrate binding site specificity of thrombin to small polar P1' residues by occluding the S1' binding pocket, based on the X-ray crystal structure of thrombin. To test this proposal, we prepared a Lys-->Ala (K60fA) mutant of recombinant thrombin and determined whether this mutation enhanced the reactivity of thrombin with a variant inhibitor [antithrombin (AT)-Denver] and a substrate (protein C) containing poorly recognized P1' Leu residues. AT-Denver in the presence of heparin inhibited K60fA thrombin with a second-order association rate constant [k = 4.2 +/- 0.1) x 10(5) M-1 s-1] that was 3.2-fold faster than thrombin [k = (1.3 +/- 0.1) x 10(5) M-1 s-1]. Wildtype AT (P1' Ser) under the same conditions inhibited K60fA thrombin with a 2.5-fold slower rate constant [k = (1.1 +/- 0.1) x 10(7) M-1 s-1] than thrombin [k = (2.8 +/- 0.1) x 10(7) M-1 s-1]. These results indicate an overall 8.3-fold improvement in the recognition of the P1' Leu of AT-Denver by K60fA thrombin over that of wild-type thrombin; i.e., the K60fA mutation partly overcomes the defect in thrombin inhibition produced by the P1' mutation in AT-Denver. Resolution of the two-step reactions of AT and AT-Denver with wild-type and mutant thrombins revealed that the enhanced recognition of P1' Leu in AT-Denver by K60fA thrombin occurs primarily in the second reaction step in which a noncovalent AT-thrombin encounter complex is converted to a stable, covalent complex. Thrombin K60fA activated Gla-domainless protein C (GDPC) approximately 2- and approximately 4-fold faster than thrombin in the presence and absence of thrombomodulin (TM), respectively, consistent with an improved interaction of the Leu P1' residue with the mutant S1' pocket. In contrast, the mutant thrombin clotted fibrinogen (P1' Gly) approximately 3-fold slower than thrombin. Kinetic analysis revealed that the improvement in the catalytic rate of activation of GDPC by K60fA thrombin in the presence of TM was localized in the second reaction step, as reflected by an approximately 2-fold increase in kcat. Direct binding studies showed that the K60fA mutation minimally affected the affinity of thrombin for Na+, indicating that the changes in S1' site-specificity of K60fA thrombin did not result from altering the allosteric transition induced by Na+. We conclude that Lys60f limits the P1' substrate and inhibitor specificity of thrombin by influencing the size and polarity of the S1' site which thereby affects the stability of the transition state for cleavage of the scissile bond in the second reaction step.
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PMID:Contribution of lysine 60f to S1' specificity of thrombin. 903 92

Activators of the sigma54-holoenzyme catalyze the isomerization of closed complexes between this polymerase and a promotor to open complexes in a reaction that depends upon hydrolysis of a nucleoside triphosphate. The activators normally bind to DNA sites with the properties of transcriptional enhancers and contact the polymerase by means of DNA loop formation. Here, we demonstrate that mutant forms of the activator nitrogen regulatory protein C (NtrC) that lack one helix of the helix-turn-helix (HTH) DNA-binding motif or the entire motif retain residual capacity to activate transcription from solution, despite the fact that they are largely unable to dimerize and have greatly decreased ability to hydrolyze ATP. We show that substitution of alanine for three hydrophilic residues in the second helix of the HTH yields a stable, dimeric form of NtrC defective in DNA-binding. Like mutant forms with deletions of one or both helices, the NtrC3ala protein failed to bind DNA in a sensitive affinity co-electrophoresis assay, indicating that its affinity for a strong enhancer was reduced by at least 5000-fold. (The assay detected enhancer-binding by two mutant forms of NtrC with single amino acid substitutions in the HTH and non-specific DNA-binding by the wild-type protein.) The phosphorylated NtrC3ala protein had normal ATPase activity in solution but, unlike the activity of the phosphorylated wild-type protein, which could be stimulated at least tenfold by an oligonucleotide carrying a strong enhancer, the ATPase activity of the phosphorylated NtrC3ala protein was not stimulated. At concentrations of 100 nM or greater, the phosphorylated NtrC3ala protein activated transcription from the major glnA promoter. In agreement with the fact that it did not show detectable DNA-binding in other assays, its ability to activate transcription was no greater on templates carrying the glnA enhancer than on templates lacking an enhancer. The results indicate that both roles of the glnA enhancer, tethering and facilitation of the formation of an active oligomer of NtrC, can be bypassed if the protein is present at high concentrations in solution.
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PMID:Mutant forms of the enhancer-binding protein NtrC can activate transcription from solution. 909 4

Mutation of three Arg residues, 93, 97, and 101, to Ala in thrombin (thrombin R93,97,101A) has previously been shown to eliminate most heparin acceleration of thrombin inhibition by antithrombin and most of the ability of chondroitin sulfate (CS) on thrombomodulin (TM) to enhance affinity for TM and to eliminate the characteristic high-affinity interaction with protein C observed with TM lacking CS. In this study we examined the relative impact of mutation of these Arg residues alone and in combination on the above reactions and, in addition, on the ability of rabbit TM to accelerate thrombin inhibition by antithrombin. The order of importance for heparin acceleration of inhibition by antithrombin was Arg 101, 93, and 97. In contrast, Arg 97 was the major residue required for TM-dependent acceleration of reactivity with antithrombin and for CS-dependent enhancement of TM affinity. Arg 101 and 93 were critical for TM-dependent, high-affinity protein C interaction at low Ca2+ concentrations, while Arg 97, which was critical for the other TM-dependent effects, played no detectable role in this metal dependence. These results illustrate that these Arg residues in anion binding exosite 2 contribute very differently to the diverse reactions dependent on that domain in thrombin.
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PMID:Influence of Arginines 93, 97, and 101 of thrombin to its functional specificity. 922 Sep 85

Factor Va (fVa) is inactivated by activated protein C (APC) by cleavage of the heavy chain at Arg306, Arg506, and Arg679. Site-directed mutagenesis of human factor V cDNA was used to substitute Arg306-->Ala (rfVa306A) and Arg506-->Gln (rfVa506Q). Both the single and double mutants (rfVa306A/506Q) were constructed. The activation of these procofactors by alpha-thrombin and their inactivation by APC were assessed in coagulation assays using factor V-deficient plasma. All recombinant and wild-type proteins had similar initial cofactor activity and identical activation products (a factor Va molecule composed of light and heavy chains). Inactivation of factor Va purified from human plasma (fVaPLASMA) in HBS Ca2+ +0.5% BSA or in conditioned media by APC in the presence of phospholipid vesicles resulted in identical inactivation profiles and displayed identical cleavage patterns. Recombinant wild-type factor Va (rfVaWT) was inactivated by APC in the presence of phospholipid vesicles at an overall rate slower than fVaPLASMA. The rfVa306A and rfVa506Q mutants were each inactivated at rates slower than rfVaWT and fVaPLASMA. Following a 90-min incubation with APC, rfVa306A and rfVa506Q retain approximately 30-40% of the initial cofactor activity. The double mutant, rfVa306A/506Q, was completely resistant to cleavage and inactivation by APC retaining 100% of the initial cofactor activity following a 90-min incubation in the presence of APC. Recombinant fVaWT, rfVa306A, rfVa506Q, and rfVa306A/506Q were also used to evaluate the effect of protein S on the individual cleavage sites of the cofactor by APC. The initial rates of rfVaWT and rfVa306A inactivation in the presence of protein S were unchanged, indicating cleavage at Arg506 is not affected by protein S. The initial rate of rfVa506Q inactivation was increased, suggesting protein S slightly accelerates the cleavage at Arg306. Overall, the data demonstrate high specificity with respect to cleavage sites for APC on factor Va and demonstrate that cleavages of the cofactor at both Arg306 and Arg506 are required for efficient factor Va inactivation.
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PMID:The effect of Arg306-->Ala and Arg506-->Gln substitutions in the inactivation of recombinant human factor Va by activated protein C and protein S. 930 May 1

The presentation pathways followed by DR1-restricted determinants from the fusion protein of measles virus were studied. By assessing the capacity of various APC preparations to stimulate fusion protein-specific T cells, it was shown that the determinant contained within the fusion protein sequence 254-268 (F254) relies on newly synthesized DR1 protein for its presentation. By contrast, the determinant contained within the fusion protein sequence 314-328 (F314) is captured by DR1 protein recycled from the plasma membrane. In vitro binding analyses showed that the F254 and F314 peptides optimally bind to DR1 at pH 4 and pH 5, respectively. In addition, it was found that binding of the F254 peptide to DR1 is much poorer at pH 7 than at pH 4, while binding of the F314 peptide was decreased only moderately at pH 7 as compared with pH 5. Substitution of the glutamic acid 261 for an alanine in the F254 peptide resulted in an analogue with an improved capacity of binding to DR1 at neutral pH. By contrast, replacement of the valine 319 by a glutamic acid in the F314 peptide generated an analogue with a decreased binding capacity at pH 7. These findings indicated that determinants that do not bear acidic residues are captured efficiently by DR1 molecules over a broader range of pH than determinants containing acidic residues. Binding analyses between DR1 and four additional peptides further supported this conclusion. Altogether, these results suggested that acidic residues, by tuning the optimal pH for the assembly of peptide-DR1 complexes, determine the processing pathway followed by the determinants.
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PMID:The processing routes determined by negatively charged residues in DR1-restricted T cell determinants. 931 22

Variant proteins containing charge-to-alanine mutations of single amino acid residues and clusters of such groups contained in the epidermal growth factor 1 (EGF1) homology unit of human protein C (PC) have been accomplished, resulting in the following recombinant (r) mutant proteins: r-[E56A/H57A]PC; r-[H66A]PC; r-[D71A]PC; r-[D79A/R81A]PC; r-[E85A/R87A]PC; and r-[R91A/E92A]PC. Studies of the mutant proteins with a variety of Ca2+-dependent and Ca2+-independent monoclonal antibodies not only led to identification of the epitopes of these antibodies, but also confirmed the importance of D/beta-hydroxyaspartic acid (Hya)71 as one probable coordination site for Ca2+. Employing these antibodies, it was also revealed that Ca2+ binding to its site in the EGF1 region of PC did not influence Ca2+ binding or adoption of the Ca2+-dependent conformation of the gamma-carboxyglutamic acid domain of this same protein. In addition, the Ca2+-induced inhibition of PC activation by thrombin, and the kinetic constants for activation of PC by the thrombin/thrombomodulin complex, were only modestly affected by any of the mutations. The mutants r-[E56A/H57A]APC and r-[H66A]APC displayed at least 70% of wild type r-APC activity in a fVIII inactivation assay, while r-[D79A/R81A]APC, r-[E85A/R87A]APC and r-[R91A/E92A]APC possessed only approximately 40% activity in that same assay. The special role of D/Hya71 in this process was confirmed by showing that r-[D71A]APC was inactive in the fVIII-inactivation assay. These findings demonstrate that some of the charged residues of EGF1, most notably those in the carboxy-terminal region of this domain, participate as partial determinants of the anticoagulant activity of APC. Overall, with the exceptions noted, the data generally suggest that the charged residues of the EGF1 domain of PC, and the Ca2+ binding site contained within this module, are likely more involved with maintenance of the overall structural integrity of this module rather than with its direct functional interactions with effectors, activators, or substrates of PC and APC. Lastly, functional Ca2+ binding to the Gla domain of PC is not significantly influenced by the binding of Ca2+ to the EGF1 module.
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PMID:The functions of the first epidermal growth factor homology region of human protein C as revealed by a charge-to-alanine scanning mutagenesis investigation. 946 48

Factor Va, the essential cofactor for prothrombinase, is phosphorylated on the acidic COOH terminus of the heavy chain of the cofactor, at Ser692, by a platelet membrane-associated casein kinase II (CKII). Consistent with this observation, phosphorylation of the factor Va heavy chain by the platelet kinase was inhibited by heparin. The membrane-associated platelet CKII kinase was partially purified using heparin-agarose, phosphocellulose, and ion exchange chromatography. CKII antigen was monitored using a polyclonal antibody to the alpha-subunit, and kinase activity in the various fractions was confirmed using human factor Va as a substrate. Immunoblotting experiments using polyclonal antibodies raised against synthetic peptides mimicking a portion of the deduced amino acid sequence of the alpha-, alpha'-, and beta-subunits of human CKII demonstrated the coexistence of both alpha- and alpha'-subunits in platelets and suggested that the platelet CKII kinase may exist in part as an alpha alpha'beta2 complex. It is also possible that there are two distinct populations of CKII in platelets, one that is alphaalpha/betabeta and one that is alpha'alpha'/betabeta. In the presence of the purified platelet-derived CKII, human factor Va incorporates between 0.8 and 1.3 mol of phosphate/mol of factor Va depending on the concentration of the beta-subunit in the kinase preparation. A peptide mimicking the sequence 687-705 of the human factor V molecule incorporates radioactivity in the presence of purified CKII and inhibits factor Va heavy chain phosphorylation by the platelet CKII. In contrast, a peptide with an alanine instead of a serine at position 692 neither incorporates phosphate nor inhibits factor Va phosphorylation by the platelet CKII. Human factor Va is inactivated by activated protein C following three cleavages of the heavy chain at Arg506, Arg306, and Arg679. Cleavage at Arg506 is necessary for efficient exposure of the inactivating cleavage site at Arg306. The phosphorylated cofactor has increased susceptibility to inactivation by activated protein C, since phosphorylated factor Va was found to be inactivated approximately 3-fold faster than its native counterpart. Acceleration of the inactivation process of the phosphorylated cofactor occurs because of acceleration of the rate of cleavage at Arg506. These data suggest a critical role for factor Va phosphorylation on the surface of platelets in regulating cofactor activity.
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PMID:Identification and partial characterization of factor Va heavy chain kinase from human platelets. 952 59

Protein C inhibitor (PCI) is a heparin-binding serine protease inhibitor (serpin) that regulates hemostatic proteases such as activated protein C (APC) and thrombin. The work described here provides further evidence that the PCI H helix, but not the D helix, has a major role in heparin-accelerated inhibition of APC and thrombin. We previously identified Arg-269 and Lys-270 of the H helix [R269A/K270A "H1" recombinant PCI (rPCI)] as important residues both for heparin-accelerated inhibition of thrombin and APC and for heparin-Sepharose binding (Shirk, R. A., Elisen, M. G. L. M., Meijers, J. C. M., and Church, F. C. (1994) J. Biol. Chem. 269, 28690-28695). H1 rPCI was used as a template for Ala-scanning mutagenesis of other H helix basic residues (H1-K266A, H1-K273A, and H1-K266A/K273A) and of the D helix basic residues (H1-K82A, H1-K86A, H1-R90A, and H1-K82A/K86A/R90A). Compared to wild-type rPCI/heparin (k2 = 2.2 x 10(7) M-1 min-1 for thrombin), heparin-accelerated thrombin inhibition was decreased 2.4-fold by H1 rPCI, 4.4-fold by H1-K266A rPCI, and 8-fold by H1-K273A rPCI. H1-K266A/K273A rPCI thrombin inhibition was essentially not accelerated by heparin. A similar trend was found for APC-heparin inhibition using these H helix rPCI mutants. In contrast, the D helix rPCI mutants did not have further reduced heparin-stimulated thrombin or APC inhibition compared to H1 rPCI. Interestingly, all of the H and D helix rPCI mutants had reduced heparin-Sepharose binding activity (ranging from 180 to 360 mM NaCl) compared to wild-type rPCI and H1 rPCI, which eluted at 650 and 430 mM NaCl, respectively. These data suggest that all four basic residues (Lys-266, Arg-269, Lys-270, Lys-273) in the H helix of PCI form a heparin binding site. Our results also imply that while the D helix basic residues (Lys-80, Lys-86, and Arg-90) contribute to overall heparin binding, they are not necessary for heparin-accelerated activity. We conclude that the primary heparin binding site of PCI is the H helix and not the D helix as found in other homologous heparin-binding serpins such as antithrombin III, heparin cofactor II, and protease nexin 1.
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PMID:Contribution of basic residues of the D and H helices in heparin binding to protein C inhibitor. 964 72

We present the protein C gene analysis of a patient with homozygous protein C deficiency. The patient was referred with purpura fulminans 3 h after birth. Skin necroses had developed on the scalp, abdomen and upper extremities when he was two days old. His protein C activity was 0.03-0.05 IU/ml and the levels in his consanguineous parents were 0.32 and 0.40 IU/ml. He was treated initially with fresh frozen plasma, and later with daily oral anticoagulants, for two episodes of skin necrosis. He had three more episodes of skin necroses that were treated with intravenous protein C concentrate. He is now two years old and under treatment with daily coumarin 0.1-0.2 mg/kg per day to keep the International Normalized Ratio between 2.0-4.5. DNA analysis showed that he is homozygous for a double variant bearing His 202 to Tyr and Ala 346 to Thr mutations. His parents were each heterozygous for the double variant and were consanguineous. This mutation has been reported previously in an Austrian patient but this is the first homozygous case for this double variant.
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PMID:Homozygous protein C deficiency with a double variant His 202 to Tyr and Ala 346 to Thr. 969 Aug 6

Epitope mapping of outer surface protein C (OspC) by using sera from patients with neuroborreliosis led to the identification of one single major immunodominant epitope within the C-terminal 10 amino acid residues. Peptide binding studies and alanine replacement scanning of the C-terminal decapeptide, PVVAESPKKP, revealed a critical role for the PKKP sequence and its terminal carboxyl group for the binding of immunoglobulin M (IgM) antibodies from patients with Lyme borreliosis. Electron microscopy of antibody-labeled spirochetes indicated that the C-terminal region is exposed on the surface of the spirochete. Based on homology to proteins of known function, this region most probably adopts a polyproline II-like helix, which is found in surface-exposed structures involved in protein-protein interactions. This structural motif is highly conserved in Borrelia species causing Lyme borreliosis and subjected to purifying selection. We suggest that the abundance of the C-terminal region of OspC on the surface of B. burgdorferi allows a multimeric high-avidity interaction between the spirochete and surface Igs on B cells. The resulting cross-linking of surface Igs on B cells may induce a T-cell-independent B-cell activation without IgM-to-IgG switching, thus explaining the lack of IgG antibodies to OspC in neuroborreliosis.
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PMID:The dominant epitope of Borrelia garinii outer surface protein C recognized by sera from patients with neuroborreliosis has a surface-exposed conserved structural motif. 971 50


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