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

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Query: EC:3.4.21.4 (trypsin)
42,187 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The contribution of a covalent bond to the stability of complexes of serine proteinases with inhibitors of the serpin family was evaluated by comparing the affinities of beta-trypsin and the catalytic serine-modified derivative, beta-anhydrotrypsin, for several serpin and non-serpin (Kunitz) inhibitors. Kinetic analyses showed that anhydrotrypsin had little or no ability to compete with trypsin for binding to alpha 1-proteinase inhibitor (alpha 1PI), plasminogen activator inhibitor 1 (PAI-1), antithrombin (AT), or AT-heparin complex when present at up to a 100-fold molar excess over trypsin. By contrast, equimolar levels of anhydrotrypsin blocked trypsin binding to non-serpin inhibitors. Equilibrium binding studies of inhibitor-enzyme interactions monitored by inhibitor displacement of the fluorescence probe, p-aminobenzamidine, from the enzyme active site, confirmed that the binding of serpins to anhydrotrypsin was undetectable in the case of alpha 1PI or AT (KI > 10(-5) M), of low affinity in the case of AT-heparin complex (KI 7-9 x 10(-6) M), and of moderate affinity in the case of PAI-1 (KI 2 x 10(-7) M). This contrasted with the stoichiometric high affinity binding of the serpins to trypsin as well as of the non-serpin inhibitors to both trypsin and anhydrotrypsin. Maximal KI values for serpin-trypsin interactions of 1 to 8 x 10(-11) M, obtained from kinetic analyses of association and dissociation rate constants, indicated that the affinity of serpins for trypsin was minimally 4 to 6 orders of magnitude greater than that of anhydrotrypsin. Anhydrotrypsin, unlike trypsin, failed to induce the characteristic fluorescence changes in a P9 Ser-->Cys PAI-1 variant labeled with a nitrobenzofuran fluorescent probe (NBD) which were shown previously to report the serpin conformational change associated with active enzyme binding. These results demonstrate that a covalent interaction involving the proteinase catalytic serine contributes a major fraction of the binding energy to serpin-trypsin interactions and is essential for inducing the serpin conformational change involved in the trapping of enzyme in stable complexes.
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PMID:Role of the catalytic serine in the interactions of serine proteinases with protein inhibitors of the serpin family. Contribution of a covalent interaction to the binding energy of serpin-proteinase complexes. 853 Apr 3

A critical component in the regulation of thrombus formation and clearance is the balance between tissue plasminogen activator (tPA) and plasminogen activator inhibitor type-1 (PAI-1). An increase in the plasma concentration of PAI-1 has been proposed as a risk factor in thrombotic disease. Inhibition of PAI-1 activity may have utility in the treatment of thromboembolic disease. We report here the evaluation of three diketopiperazine-based low molecular weight inhibitors of PAI-1 activity (XR334, XR1853 and XR5082). In vitro these compounds reversed the inhibitory effects of PAI-1 against both tPA and urokinase (UK) (IC50: 5 to 80 muM). In contrast, other serpin-serine protease interactions, including alpha 1-antitrypsin-trypsin, alpha 2-antiplasmin- plasmin and antithrombin-thrombin, were not affected, neither did these inhibitors affect global tests of haemostasis. In the light of this promising in vitro profile these compounds were evaluated in a standard radioisotopic assay of clot lysis in whole rat blood following intravenous administration. In this assay these compounds dose-dependently enhanced fibrinolysis ex vivo. After intravenous bolus administration XR334, XR1853 and XR5082 at 5 mg/kg increased clot lysis by 32.0 +/- 5.1% SEM (n = 25, p < 0.01), 36.7 +/- 3.5% SEM (n = 36, p < 0.01) and 60.0 +/- 2.8% SEM (n = 17, p < 0.01) respectively compared to vehicle. Intravenous infusion of these compounds (1 mg/kg/min for 20 min) significantly prolonged (approximately twofold) the time to blood vessel occlusion in the rat electrically-stimulated carotid artery thrombosis model. Thus, these low molecular weight inhibitors of PAI-1 activity enhanced fibrinolysis ex vivo and protected against thrombus formation in the rat.
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PMID:Evaluation of a low molecular weight modulator of human plasminogen activator inhibitor-1 activity. 872 28

Quantitative characterization of the interaction of des-kringle1-5-plasmin (microplasmin) with fibrin(ogen) and plasma protease inhibitors may serve as a tool for further evaluation of the role of kringle domains in the regulation of fibrinolysis. Comparison of fibrin(ogen) degradation products yielded by plasmin, miniplasmin (des-kringle1-4-plasmin), microplasmin, and trypsin on SDS gel electrophoresis indicates that the differences in the enzyme structure result in different rates of product formation, whereas the products of the four proteases are very similar in molecular weight. Kinetic parameters show that plasmin is the most efficient enzyme in fibrinogen degradation, and the kcat/KM ratio decreases in parallel with the loss of the kringle domains. The catalytic sites of the four proteases have similar affinities for fibrin (KM values between 0.12 and 0.21 microM). Trypsin has the highest catalytic constant for fibrin digestion (kcat = 0.47 s-1), and among plasmins with different kringle structures, the loss of kringle5 results in a markedly lower catalytic rate constant (kcat = 0.0076 s-1 for microplasmin vs 0.048 s-1 for miniplasmin and 0.064 s-1 for plasmin). In addition, microplasmin is inactivated by plasmin inhibitor (k" = 3.9 x 10(5) M-1 s-1) and antithrombin (k" = 1.4 x 10(3) M-1 s-1) and the rate of inactivation decreases in the presence of fibrin(ogen). Heparin (250 nM) accelerates the inactivation of microplasmin by antithrombin (k" = 10.5 x 10(3) M-1 s-1 ), whereas that by plasmin inhibitor is not affected (k" = 4.2 x 10(5) M-1 s-1).
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PMID:Fibrinolysis with des-kringle derivatives of plasmin and its modulation by plasma protease inhibitors. 963 57

Most secretory proteins, including antithrombin (AT), are synthesized with a signal peptide, which is cleaved before the mature protein is exported from the cell. The signal peptide is important in the process whereby nascent protein is recognized as requiring subsequent modification within the endoplasmic reticulum (ER). We have identified a novel mutation, 2436T-->C L(-10)P, which affects the central hydrophobic domain of the AT signal peptide, in a proband presenting with venous thrombotic disease and type I AT deficiency. We investigated the basis of the phenotype by examining expression in mammalian cells of a range of variant AT cDNAs with mutations affecting the -10 residue. Glycosylated AT was secreted from COS-7 cells transfected with wild-type AT, -10L deletion, -10V or -10M variants, but not variants with P, T, R, or G at -10. Cell-free expression of wild-type and variant AT cDNAs was then performed in the presence of canine pancreatic microsomes, as a substitute for ER. Variant AT proteins with P, T, R, or G at residue -10 did not undergo posttranslational glycosylation, and their susceptibility to trypsin digestion suggested they had not been translocated into microsomes. Our results suggest that the ability of AT signal peptide to direct the protein to ER for cotranslational processing events appears to be critically dependent on maintaining the hydrophobic nature of the region including residue -10. The investigations have defined impaired cotranslational processing as a hitherto unrecognized cause of hereditary AT deficiency.
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PMID:Impaired cotranslational processing as a mechanism for type I antithrombin deficiency. 984 33

Eight patients with pancreatic abscesses secondary to acute necrotizing pancreatitis underwent drainage of their abscesses under laparotomy. Two of them died of acute pulmonary thromboembolism (PTE) within 1 week. Autopsy revealed a large thrombus at the main trunk of the pulmonary artery and in the left common iliac vein. Femoral catheter insertion/indwelling, immobilization, surgery, increased trypsin/kinin/kallikrein, increased endotoxin, and decreased antithrombin-III (AT-III) were present following drainage of the pancreatic abscesses. With respect to the bedside diagnosis of acute PTE, alveolar-arterial oxygen gradients obtained by blood gas analysis and mean pulmonary artery pressure estimated by pulsed Doppler echocardiography are very useful. In terms of the treatment, attention should be paid to the following to prevent deep venous thrombosis: prophylactic administration of low molecular weight heparin and administration of AT-III (AT-III > or = 80%), use of the subclavian vein whenever possible as blood access for apheresis therapy, as short a compression time as possible after removing the blood access catheter (< or =6 h), and application of intermittent pneumatic compression devices or elastic compression stockings on the lower extremities.
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PMID:Postoperative acute pulmonary thromboembolism in patients with acute necrotizing pancreatitis with special reference to apheresis therapy. 1022 70

Based on the structural comparison of the S-1 pocket in different trypsin-like serine proteases, a series of Boc-D-trimethylsilylalanine-proline-boro-X pinanediol derivatives, with boro-X being different amino boronic acids, have been synthesised as inhibitors of thrombin. The influence of hydrogen donor/acceptor properties of different residues in the P-1 side chain of these inhibitors on the selectivity profile has been investigated. This study confirmed the structure-based working hypothesis: The hydrophobic/hydrophilic character of amino acid residues 190 and 213 in the neighbourhood of Asp 189 in the S-1 pocket of thrombin (Ala/Val), trypsin (Ser/Val) and plasmin (Ser/Thr) define the specificity for the interaction with different P-1 residues of the inhibitors. Many of the synthesised compounds demonstrate potent antithrombin activity with Boc-D-trimethylsilylalanine-proline-boro-methoxypropylglycine++ + pinanediol (9) being the most selective thrombin inhibitor of this series.
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PMID:Design, synthesis and biological evaluation of selective boron-containing thrombin inhibitors. 1046 5

From the bloodsucking bug Dipetalogaster maximus, a protein with anticoagulant activity was isolated and biochemically characterized. The isolated protein, named dipetalogastin, possesses an average molecular mass of 11.8 kD. Its N-terminal sequence shows homology to rhodniin, a thrombin inhibitor isolated from the bug Rhodnius prolixus. The in vitro anticoagulant activity of dipetalogastin occurs via the inhibition of thrombin. The anticoagulant and thrombin inhibitory potency of dipetalogastin is comparable to that of recombinant hirudin. Its specific thrombin inhibitory activity is 9,300 antithrombin units/mg protein. Dipetalogastin forms only 1:1 molar complexes with thrombin. It is a tight-binding inhibitor of thrombin possessing a dissociation constant of 125 fM. It does not inhibit factor Xa or alpha-chymotrypsin and only weakly inhibits trypsin.
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PMID:Biochemical characterization of a thrombin inhibitor from the bloodsucking bug Dipetalogaster maximus. 1070 1

A polyclonal anti-bovine pancreatic trypsin inhibitor (BPTI) IgY was raised in chickens immunised with aprotinin. The anti-BPTI IgY was subsequently isolated from egg yolks and purified to homogeneity by affinity chromatography on immobilised aprotinin and by Superose 6 size exclusion fast protein liquid chromatography (FPLC). Immunoblotting with the chicken IgY demonstrated its specificity for BPTI; 3.9 ng BPTI could be detected by this technique. There was no crossreactivity against alpha1-proteinase inhibitor (human and sheep), inter-alpha-trypsin inhibitor (human and sheep), secretory leucocyte proteinase inhibitor or a range of serine proteinase inhibitory proteins (SPIs) isolated from plant sources (soybean and lima bean trypsin inhibitor, potato trypsin and chymotrypsin inhibitors) or serum SPIs (antithrombin-III, alpha2-macroglobulin). Immunoblotting using the anti-BPTI IgY identified the 6- to 12- and 58-kDa forms of endogenous ovine cartilage SPIs in cartilage extracts, confirming the interrelationship of the ovine cartilage SPIs with BPTI. BPTI-domain SPIs were immunolocalised within mast cells of ovine and bovine duodenum, lung and pancreas, and in ovine and bovine bronchial cartilage chondrocytes, chondrocytes of the superficial and intermediate zones of articular cartilage and in the fibrochondrocytes/chondrocytes of the nucleus
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PMID:Immunolocalisation of BPTI-like serine proteinase inhibitory proteins in mast cells, chondrocytes and intervertebral disc fibrochondrocytes of ovine and bovine connective tissues. An immunohistochemical and biochemical study. 1105 62

Heparin has been proposed to conformationally activate the serpin, antithrombin, by making the reactive center loop P1 arginine residue accessible to proteinases. To evaluate this proposal, we determined the effect of mutating the P1 arginine on antithrombin's specificity for target and nontarget proteinases in both native and heparin-activated states of the serpin. As expected, mutation of the P1 arginine to tryptophan, histidine, leucine, and methionine converted the specificity of antithrombin from a trypsin inhibitor (k(assoc) = 2 x 10(5) M(-1) s(-1)) to a chymotrypsin inhibitor (k(assoc) = 10(3)-10(5) M(-1) s(-1)). However, heparin pentasaccharide activation increased the reactivity of the P1 variants with chymotrypsin or of the wild-type inhibitor with trypsin only 2-6-fold, implying that the P1 residue had similar accessibilities to these proteinases in native and activated states. Mutation of the P1 arginine greatly reduced k(assoc) for antithrombin inhibition of thrombin and factor Xa from 40- to 5000-fold, but heparin normally accelerated the reactions of the variant antithrombins with these enzymes to make them reasonably efficient inhibitors (k(assoc) = 10(3)-10(4) M(-1) s(-1)). Fluorescence difference spectra of wild-type and P1 tryptophan variant antithrombins showed that the P1 tryptophan exhibited fluorescence properties characteristic of a solvent-exposed residue which were insignificantly affected by heparin activation. Moreover, all P1 variant antithrombins bound heparin with approximately 2-3-fold higher affinities than the wild type. These findings are consistent with the P1 mutations disrupting a P1 arginine-serpin body interaction which stabilizes the native low-heparin affinity conformation, but suggest that this interaction is of low energy and unlikely to limit the accessibility of the P1 residue. Together, these findings suggest that the P1 arginine residue is similarly accessible to proteinases in both native and heparin-activated states of the serpin and contributes similarly to the specificity of antithrombin for thrombin and factor Xa in the two serpin conformational states. Consequently, determinants other than the P1 residue are responsible for enhancing the specificity of antithrombin for the two proteinases when activated by heparin.
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PMID:The antithrombin P1 residue is important for target proteinase specificity but not for heparin activation of the serpin. Characterization of P1 antithrombin variants with altered proteinase specificity but normal heparin activation. 1138 Feb 62

When active serpins are proteolytically inactivated in a substrate-like reaction, they undergo an important structural transition with a resultant increase in their conformational stability. We have used microcalorimetry to show that this conformational alteration is accompanied by an important enthalpy change. For instance, the cleavage of alpha(1)-proteinase inhibitor by Pseudomonas aeruginosa elastase, Staphylococcus aureus V8 proteinase, or papain and that of antithrombin by leukocyte elastase are characterized by large enthalpy changes (DeltaH = -53 to -63 kcal mol(-1)). The former reaction also has a large and negative heat capacity (DeltaC(p)() = -566 cal K(-1) mol(-1)). In contrast, serpins release significantly less heat when they act as proteinase inhibitors. For example, the inhibition of pancreatic elastase, leukocyte elastase, and pancreatic chymotrypsin by alpha(1)-proteinase inhibitor and that of pancreatic trypsin and coagulation factor Xa by antithrombin are accompanied by a DeltaH of -20 to -31 kcal mol(-1). We observe no heat release upon proteolytic cleavage of inactive serpins or following inhibition of serine proteinases by canonical inhibitors or upon acylation of chymotrypsin by N-trans-cinnamoylimidazole. We suggest that part of the large enthalpy change that occurs during the structural transition of serpins is used to stabilize the proteinase in its inactive state.
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PMID:The reaction of serpins with proteinases involves important enthalpy changes. 1150 92


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