Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

---

Query: HUMANGGP:027518 (factor Xa)
5,167 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Tick anticoagulant peptide (TAP) is a specific and potent inhibitor of factor Xa (fXa), a central enzyme in the blood clotting cascade. As such, TAP is a potential antithrombotic agent. Site-directed mutagenesis studies were undertaken to determine the feasibility of increasing the inhibitory potency of TAP toward fXa. The amino acid substitutions Tyr-1 to Trp (Y1W) and Asp-10 to Arg (D10R) increased inhibitory potency toward human fXa by 2.5- and 4-fold, respectively. The increased inhibitory potency reflected a decrease in the rate constant for dissociation of the final fXa-TAP inhibitory complex. The double mutant, Y1W/D10R, exhibited an inhibition constant of 10 pM, a 37-fold enhancement of inhibitory potency toward human fXa. The improvement in inhibitory potency was less pronounced (12-fold) with dog fXa wherein Kis of 220 and 18 pM were observed for wild-type TAP and the double mutant, respectively. Mutation of Tyr-1 to Glu (Y1E) generated a weaker inhibitor (Ki = 2 nM) that bound human fXa more slowly. However, no change in inhibitory potency toward human fXa was observed when Tyr-1 was replaced by Phe. Taken together, these observations are consistent with the view that a hydrophobic amino acid at the N-terminus of TAP may be a determinant of inhibitory potency. Decreases by 3-4 orders of magnitude in inhibitory potency were noted upon mutation of Asn-2 and Leu-4 of TAP, further implicating the N-terminal domain as an important determinant of inhibitory potency.(ABSTRACT TRUNCATED AT 250 WORDS)
...
PMID:Identification and characterization of variants of tick anticoagulant peptide with increased inhibitory potency toward human factor Xa. 771 Oct 29

It has been suggested (Kini, R. R., and Evans, H. J. (1987) J. Biol. Chem. 262, 14402-14407) that the anticoagulant activity of members of the 14-kDa phospholipase A2 (PLA2) family depends on the presence of basic residues within a variable surface region (residues 54-77) distinct from both the conserved catalytic machinery and surface sites mediating the antibacterial action of these enzymes (see Weiss, J., Inada, M., Elsbach, P., and Crowl, R. M. (1994) J. Biol. Chem. 269, 26331-26337). To further define the determinants of the anticoagulant activity of PLA2, we have analyzed the inhibitory effects of purified native and recombinant PLA2 on cell-free prothrombinase. Both native and recombinant wild-type pig pancreas (net charge -1) and human "secretory" PLA2 (net charge +15) produced similar dose-dependent inhibition of prothrombinase activity that was significantly less potent than a toxic PLA2 purified from snake venom. Site-specific mutations that either increased or decreased PLA2 activity toward bactericidal/permeability-increasing protein-treated Escherichia coli by up to 50-fold had no effect on antiprothrombinase activity. In contrast, substitution of Arg for Asp-59/Gly for Ser-60 in the pig PLA2 increased antiprothrombinase activity by 5-10-fold without affecting catalytic activity toward a range of phospholipid substrates or antibacterial activity. Comparison of antiprothrombinase activity of catalytically active and inactive forms of the PLA2 and under a range of phospholipid conditions revealed that the potent antiprothrombinase activity of native toxic venom PLA2 and of the D59R.S60G mutant pancreatic PLA2 reflect combined catalytic and noncatalytic actions, the latter apparently dependent on basic residues at discrete surface sites in the enzyme.
...
PMID:Determinants of the inhibitory action of purified 14-kDa phospholipases A2 on cell-free prothrombinase complex. 792 51

Protein C is a vitamin K-dependent serine protease zymogen that upon activation inhibits the coagulation cascade by inactivating factors Va and VIIIa. In an attempt to improve the anticoagulant activity of activated protein C (APC), we have prepared a mutant of protein C in mammalian cells in which Glu at position 192 (chymotrypsin numbering system) has been replaced with Gln (PC E192Q). Our strategy is based on the observation that the same substitution in thrombin improves the catalytic activity toward natural and synthetic substrates that contain Asp residues at P3 and P3'. Since factor Va also has an Asp at position P3 in the APC cleavage site of the factor Va heavy chain, we hypothesized that APC E192Q would inactivate factor Va more rapidly than wild type APC. The mutant inactivated factor Va approximately 2-3-fold faster than wild type. In plasma the mutant exhibited slightly less anticoagulant activity than wild type enzyme. Further characterization revealed that APC E192Q is inhibited 280 times faster than APC by alpha 1-antitrypsin (K2 = 2.8 x 10(3) M-1S-1 versus 10 M-1 S-1), and unlike APC, APC E192Q is inhibited by antithrombin III in the presence of heparin (K2 = 1.17 x 10(3) M-1 S-1) M-1 S-1) and absence of heparin (K2 = 57 M-1 S-1). Ca2+ increased K2 more than 4-fold with or without heparin. Unlike wild type APC, APC E192Q was effectively inhibited by pancreatic trypsin inhibitor (Ki = 10.6 +/- 0.26 nM) and tissue factor pathway inhibitor (58 +/- 5 nM). Like factor Xa, APC E192Q rapidly processed factor IX to factor IX alpha. These observations suggest that even though Glu at position 192 is not an optimal residue for catalyzing factor Va inactivation, it is an evolutionary adaptation to slow inhibition by plasma protease inhibitors.
...
PMID:Conversion of glutamic acid 192 to glutamine in activated protein C changes the substrate specificity and increases reactivity toward macromolecular inhibitors. 810 82

The dependence of the activity of recombinant activated human protein C (r-APC) on each of its nine gamma-carboxyglutamic (Gla) residues (sequence positions 6, 7, 14, 16, 19, 20, 25, 26, and 29) has been assessed in purified systems and in plasma using r-mutants in which each Gla residue of r-APC was individually altered to an Asp (D) residue. The assays employed included a factor Va inactivation assay in the prothrombinase system with purified components and in plasma. In addition, a factor VIII inactivation assay in the tenase system, also with purified components, was utilized. Compared to wild-type protein (wtr-APC), the r-mutants that possessed nearly full activity in all assays were the Gla6-->D variant ([Gla6D]r-APC]) as well as [Gla14D]r-APC and [Gla19D]r-APC. In addition, another mutant (Q32-->Gla) in which a Gla was substituted for Gln (Q) at position 32, a situation that exists with other vitamin-K-dependent clotting proteins (e.g., factor IX and prothrombin), displayed full activity in all assays. Those mutants that possessed very-low-to-no activity in all assays included [Gla16D]r-APC and [Gla26D]r-APC. The other mutants showed partial and, in some cases, differential activity in these assay systems, with [Gla25D]r-APC being the most remarkable example. In this case, the factor V/Va plasma assay and the plasma-based activated partial thromboplastin time assay yielded < 25% activity, whereas nearly full activity was observed for this variant in the prothrombinase and tenase assays with purified components.(ABSTRACT TRUNCATED AT 250 WORDS)
...
PMID:The activities of recombinant gamma-carboxyglutamic-acid-deficient mutants of activated human protein C toward human coagulation factor Va and factor VIII in purified systems and in plasma. 811 Jul 90

Prothrombin has 10 gamma-carboxyglutamic acid residues which are essential for the metal ion binding properties and membrane binding function of the protein. To assess the importance of each gamma-carboxyglutamic acid residue we made, by site directed mutagenesis, a series of mutant human prothrombins each with a single glutamic acid to aspartic acid substitution at positions 6, 7, 14, 16, 19, 20, 25, 26, 29, or 32 which are gamma-carboxylated in native prothrombin. Along with wild-type prothrombin, the prothrombin mutants were expressed in Chinese hamster ovary cells, purified by immunoaffinity chromatography using polyclonal anti-prothrombin antibodies, and shown by amino acid analysis to contain the expected number of gamma-carboxyglutamic acid residues. Only substitution of gamma-carboxyglutamic acid 6 with aspartic acid yields a protein with procoagulant activity, affinity for phospholipid and KM(app) for prothrombinase indistinguishable from wild-type prothrombin. In contrast, the conservative gamma-carboxyglutamic acid to aspartic acid mutation at positions 16, 26, or 29 results in proteins with little or no procoagulant activity, Kd(app) for binding to phospholipid at least 200-fold higher than wild-type prothrombin and a KM(app) for interaction with the prothrombinase complex nearly 100-fold higher than wild-type prothrombin. The mutations at residues 7, 14, 19, 20, 25, or 32 yielded proteins with intermediate procoagulant activities, between 15 and 52% of wild-type prothrombin. These data have been interpreted to suggest that there are certain gamma-carboxyglutamic acid residues which are important to maintaining the basic structure of the calcium-liganded Gla domain while other gamma-carboxyglutamic acid residues subserve other functions including membrane binding and interdomain interactions.
...
PMID:The importance of specific gamma-carboxyglutamic acid residues in prothrombin. Evaluation by site-specific mutagenesis. 822 83

Antithrombin (AT) is the principal inhibitor of thrombin in human plasma, and a member of the serine proteinase (serpin) family of proteins. Previously, we have described a point mutation in the human AT gene that converted amino acid 392 from glycine to aspartic acid which was associated with thrombotic disease in a Swedish family [(1992) Blood 79, 1428-1434]. This observation prompted us to investigate the consequences of other substitutions at this position, termed P2 with respect to the reactive centre. Site-directed mutagenesis was employed to generate seven mutants (Pro, Met, Gln, Val, Lys, Glu, and Asp), whose properties were compared with wild-type recombinant AT, following in vitro transcription and cell-free expression in a rabbit reticulocyte lysate system. With only one exception, the variant forms were less active than the wild-type in forming complexes with either alpha-thrombin, factor Xa, or trypsin. Hydrophobic (Val) or negatively charged (Asp or Glu) substitutions were particularly disruptive, in that these variants exhibited less than 10% wild-type antithrombin or antitrypsin activity. In contrast, the formation of complexes with the various proteases of the Pro variant was essentially unimpaired. We conclude that the P2 residue of AT plays a role in optimal presentation of the reactive centre to its cognate protease, and propose that the observed requirement of Gly or Pro at this position is suggestive of a bend in the polypeptide backbone that aids in this presentation.
...
PMID:Site-directed mutagenesis of the P2 residue of human antithrombin. 831 64

S-peptide (residues 1-20) and S-protein (residues 21-124) are the enzymatically inactive products of the limited digestion of ribonuclease A by subtilisin. S-peptide binds S-protein with high affinity to form ribonuclease S, which has full enzymatic activity. Recombinant DNA technology was used to produce a fusion protein having three parts: carrier, spacer, and target. The two carriers used were the first 15 residues of S-peptide (S15) and a mutant S15 in which Asp 14 had been changed to Asn (D14N S15). The spacer consisted of three proline residues and a four-residue sequence recognized by factor Xa protease. The target was beta-galactosidase. The interaction between the S-peptide portion of the fusion protein and immobilized S-protein allowed for affinity purification of the fusion protein under denaturing (S15 as carrier) or nondenaturing (D14N S15 as carrier) conditions. A sensitive method was developed to detect the fusion protein after sodium dodecyl sulfate-polyacrylamide gel electrophoresis by its ribonuclease activity following activation with S-protein. S-peptide has distinct advantages over existing carriers in fusion proteins in that it combines a small size (> or = 15 residues), a tunable affinity for ligand (Kd > or = 10(-9) M), and a high sensitivity of detection (> or = 10(-16) mol in a gel).
...
PMID:Ribonuclease S-peptide as a carrier in fusion proteins. 845 73

A series of site-specific mutants of the phosphatidylcholine-preferring phospholipase C from Bacillus cereus (PLCBc) was prepared in which the glutamic acid residue at position 146 was replaced with glutamine, aspartic acid, histidine, and leucine to elucidate what role Glu146 might play in catalysis. An expression system for the native enzyme in Escherichia coli was first developed to provide PLCBc that was fused via an intervening factor Xa protease recognition sequence at its N-terminus to maltose binding protein (MBP). This MBP-PLCBc fusion protein was isolated at levels of 50-70 mg/L of culture; selective trypsin digestion of the MBP-PLCBc fusion protein followed by chromatographic purification yielded recombinant PLCBc at levels of ca. 10 mg/L. Polymerase chain reaction (PCR) mutagenesis on the PLCBc gene (plc) was then used to replace the Glu146 codon with those for glutamine (E146Q), aspartic acid (E146D), histidine (E146H), and leucine (E146L). The catalytic efficiency of the E146Q mutant was 1.6% that of native PLCBc, while the other mutants each possessed activities of 0.2-0.3% of the wild type. The kcat/Km vs pH profiles for both E146Q and native PLCBc have ascending acidic limbs, suggesting that Glu146 does not serve as the general base in the hydrolysis reaction. As measured by circular dichroism, all of the mutant proteins contained less helical structure and underwent denaturation at lower temperatures than the wild type in the order: wild type > E146Q > E146D approximately E146H approximately E146L. Atomic absorption analyses indicated that the mutant proteins also exhibited lower Zn2+ content than the wild type. Thus, the Glu146 residue in PLCBc stabilizes the secondary and tertiary structure of the enzyme and serves as a critical ligand for Zn2, but it does not appear to have any specific catalytic role.
...
PMID:Expression and site-directed mutagenesis of the phosphatidylcholine-preferring phospholipase C of Bacillus cereus: probing the role of the active site Glu146. 884 Nov 44

A unique blood coagulation factor X variant has been identified in a family with a history of bleeding. Plasma from affected family members had prolonged prothrombin times and activated partial thromboplastin times, low to below normal factor X coagulant activity, and normal factor X antigen levels. Sequencing of DNA from the propositus revealed a single G to A substitution in one allele of factor X at base 964 resulting in an amino acid substitution of Asn for Asp at residue 282. This residue corresponds with the active site Asp102 of chymotrypsin. The substitution eliminates a TaqI restriction site and provided the basis for a screening assay to detect the mutation in polymerase chain reaction (PCR) amplified factor X exon VIII DNA. Fourteen additional family members were identified as having the mutation at base 964. Plasma factor X purified from the proposita using an anti-factor X monoclonal antibody immunoadsorbent exhibited an approximately 50% decrease in specific activity compared with factor X purified from a normal individual in a similar manner. Bleeding in family members with the mutation, termed factor X Stockton, appears to be due to disruption of normal hemostasis by the presence in plasma of circulating abnormal factor X. Factor X Stockton is the first naturally occurring substitution at the active site Asp of a serine protease and underscores the importance of this amino acid residue in factor Xa coagulant activity.
...
PMID:Factor X Stockton: a mild bleeding diathesis associated with an active site mutation in factor X. 884 63

Staphylokinase (Sak) forms an inactive 1:1 stoichiometric complex with plasminogen which requires both conversion of plasminogen to plasmin and hydrolysis of the Lys10-Lys11 peptide bond of Sak to become a potent plasminogen activator (Schlott, B., Guhrs, K.-H., Hartmann, M., Rocker, A., and Collen, D. (1997) J. Biol. Chem. 272, 6067-6072). Exposure of a positively charged NH2-terminal amino acid after hydrolysis of Sak is a major determinant of the plasminogen-activating potential, but in itself is neither necessary nor sufficient. Here, the structural motifs of the NH2-terminal region Lys11-Gly-Asp-Asp-Ala-Ser16-Tyr-Phe-Glu of processed Sak, required for plasminogen activating potential, were studied by deletion and substitution mutagenesis. Expression in Escherichia coli of variants with deletion of 11, 14, 15, or 16 NH2-terminal amino acids yielded correctly processed but inactive molecules. Expression of their homologues with the NH2-terminal amino acid substituted with Lys-generated derivatives from which the NH2-terminal initiation Met was no longer removed, yielding inactive (</= 10%) Sak42DDeltaN11(M),G12K, active (>50%) Sak42DDeltaN14(M), A15K and Sak42DDeltaN15(M),S16K, and inactive Sak42DDeltaN16(M),Y17K. Lys variants without NH2-terminal Met, generated from fusion proteins in which a His6 tag and a factor Xa recognition sequence were linked to the NH2 terminus of the Sak variants, were indistinguishable from their NH2-terminal Met-containing counterparts. All variants studied had intact affinities for plasminogen as measured by biospecific interaction analysis. The activity of Sak42DDeltaN11(M),G12K could be restored by additional substitution of both Asp13 and Asp14 with Asn, yielding active Sak42DDeltaN11(M),G12K, D13N, D14N, whereas substitution in Sak42DDeltaN16(M),Y17K of Phe18 and Glu19 with Asn yielded inactive Sak42DDeltaN16(M),Y17K,F18N,E19N. These data, in combination with the recent finding that the 20 NH2-terminal amino acids of Sak lack secondary structure, suggest that the NH2-terminal region of Sak is not required for binding to plasmin/plasminogen, but that a positively charged amino acid in the ultimate or penultimate NH2-terminal position corresponding to amino acids 11-16 of this flexible region participates in the reconfiguration of the active site of the plasmin molecule to endow it with plasminogen-activating potential.
...
PMID:NH2-terminal structural motifs in staphylokinase required for plasminogen activation. 971 54


<< Previous 1 2 3 4 5 Next >>

---