Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.4.21.1 (chymotrypsin)
 10,938 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Necrotic is a member of the serine protease inhibitor or serpin superfamily. It is a potent inhibitor of elastase and chymotrypsin type proteases and is responsible for regulating the anti-fungal response in Drosophila melanogaster. Necrotic contains three basic lysine residues within the D-helix that are homologous to those found in the heparin-binding domain of antithrombin and heparin co-factor II. We show here that substitution of all three lysine residues for glutamines caused cellular necrosis and premature death in Drosophila in keeping with a loss of function phenotype. The lysine to glutamine substitutions had no effect on the overall structure of recombinant Necrotic protein but abolished the formation of stable complexes with target proteases. Individual substitutions with either glutamine or alanine demonstrated that lysine 68 was the most critical residue for inhibitory activity. Despite the homology to other serpins, Necrotic did not bind, nor was it activated by sulfated glycans. These data demonstrate a critical role for basic residues within the D-helix (and lysine 68 in particular) in the inhibitory mechanism of the serpin Necrotic.
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PMID:Inhibitory activity of the Drosophila melanogaster serpin Necrotic is dependent on lysine residues in the D-helix. 1683 44

Ecotin is a Escherichia coli-derived protein that has been characterized as a potent inhibitor of serine-proteases. This protein is highly effective against several mammalian enzymes, which includes pancreatic and neutrophil-derived elastases, chymotrypsin, trypsin, factor Xa, and kallikrein. In this work we showed that ecotin binds to human alpha-thrombin via its secondary binding site, and modulates thrombin catalytic activity. Formation of wild type ecotin-alpha-thrombin complex was observed by native PAGE and remarkably, gel filtration chromatography showed an unusual 2:1 ecotin:enzyme stoichiometry. Analysis of the protease inhibitor effects on thrombin biological activities showed that (i) it decreases the inhibition of thrombin by heparin/antithrombin complex (IC50=3.2 microM); (ii) it produces a two-fold increase in the thrombin-induced fibrinogen clotting; and (iii) it inhibits thrombin-induced platelet aggregation (IC50=4.5 microM). Allosteric changes on thrombin structure were then evaluated. Complex formation with ecotin caused a three-fold increase in the rate of thrombin inhibition by BPTI, suggesting a displacement of the enzyme's 60-loop. In addition, ecotin modulated the enzyme's catalytic site, as demonstrated by changes in the fluorescence emission of fluorescein-FPRCK-alpha-thrombin (EC50=3.5 microM). Finally, solid phase competition assays demonstrated that heparin and prothrombin fragment 2 prevents thrombin interaction with ecotin. Altogether, these observations strongly support an ecotin interaction with thrombin anion-binding exosite-2, resulting in modulation of its biological activities. At this point, ecotin might be useful as a new tool for studying thrombin allosteric modulation.
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PMID:Ecotin modulates thrombin activity through exosite-2 interactions. 1684

Tissue factor pathway inhibitor (TFPI) is a multivalent Kunitz-type protease inhibitor that primarily inhibits the extrinsic pathway of blood coagulation. It is synthesized by various cells and its expression level increases in inflammatory environments. Mast cells and neutrophils accumulate at sites of inflammation and vascular disease where they release proteinases as well as chemical mediators of these conditions. In this study, the interactions between TFPI and serine proteinases secreted from human mast cells and neutrophils were examined. TFPI inactivated human lung tryptase, and its inhibitory activity was stronger than that of antithrombin. In contrast, mast cell chymase rapidly cleaved TFPI even at an enzyme to substrate molar ratio of 1:500, resulting in markedly decreased TFPI anticoagulant and anti-(factor Xa) activities. N-terminal amino-acid sequencing and MS analyses of the proteolytic fragments revealed that chymase preferentially cleaved TFPI at Tyr159-Gly160, Phe181-Glu182, Leu89-Gln90, and Tyr268-Glu269, in that order, resulting in the separation of the three individual Kunitz domains. Neutrophil-derived proteinase 3 also cleaved TFPI, but the reaction was much slower than the chymase reaction. In contrast, alpha-chymotrypsin, which shows similar substrate specificities to those of chymase, resulted in a markedly lower level of TFPI degradation. These data indicate that TFPI is a novel and highly susceptible substrate of chymase. We propose that chymase-mediated proteolysis of TFPI may induce a thrombosis-prone state at inflammatory sites.
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PMID:Tissue factor pathway inhibitor is highly susceptible to chymase-mediated proteolysis. 1750 77

We recently demonstrated that the substitution of the autolysis loop (residues 143 to 154 in the chymotrypsin numbering system) of activated protein C (APC) with the corresponding loop of factor Xa (fXa) renders the APC mutant (APC/fX143-154) susceptible to inhibition by antithrombin (AT) in the presence of pentasaccharide. Our recent results further indicated, that in addition to an improvement in the reactivity of APC/fX143-154 with AT, both the amidolytic and anti-factor Va activities of the mutant APC have also been significantly increased. Since the autolysis loop of APC is five residues longer than the autolysis loop of fXa, it could not be ascertained whether this loop in the mutant APC specifically interacts with the activated conformation of AT or if a shorter autolysis loop is responsible for a global improvement in the catalytic activity of the mutant protease. To answer this question, we prepared another APC mutant in which the autolysis loop of the protease was replaced with the corresponding loop of trypsin (APC/Tryp143-154). Unlike an approximately 500-fold improvement in the reactivity of APC/fX143-154 with AT in the presence of pentasaccharide, the reactivity of APC/Tryp143-154 with the serpin was improved approximately 10-fold. These results suggest that both the length and structure of residues of the autolysis loop are critical for the specificity of the coagulation protease interaction with AT. Further factor Va inactivation studies with the APC mutants revealed a similar role for the autolysis loop of APC in the interaction with its natural substrate.
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PMID:The role of autolysis loop in determining the specificity of coagulation proteases. 1766 41

In serine proteases, Gly193 (chymotrypsin numbering) is conserved with rare exception. Mutants of blood coagulation proteases have been reported with Glu, Ala, Arg or Val substitutions for Gly193. To further understand the role of Gly193 in protease activity, we replaced it with Ala or Val in coagulation factor XIa (FXIa). For comparison to the reported FXIa Glu193 mutant, we prepared FXIa with Asp (short side chain) or Lys (opposite charge) substitutions. Binding of p-aminobenzamidine (pAB) and diisopropylfluorphosphate (DFP) were impaired 1.6-36-fold and 35-478-fold, respectively, indicating distortion of, or altered accessibility to, the S1 and oxyanion-binding sites. Val or Asp substitutions caused the most impairment. Salt bridge formation between the amino terminus of the mature protease moiety at Ile16 and Asp194, essential for catalysis, was impaired 1.4-4-fold. Mutations reduced catalytic efficiency of tripeptide substrate hydrolysis 6-280-fold, with Val or Asp causing the most impairment. Further studies were directed toward macromolecular interactions with the FXIa mutants. kcat for factor IX activation was reduced 8-fold for Ala and 400-1100-fold for other mutants, while binding of the inhibitors antithrombin and amyloid beta-precursor protein Kunitz domain (APPI) was impaired 13-2300-fold and 22-27000-fold, respectively. The data indicate that beta-branching of the side chain of residue 193 is deleterious for interactions with pAB, DFP and amidolytic substrates, situations where no S2'-P2' interactions are involved. When an S2'-P2' interaction is involved (factor IX, antithrombin, APPI), beta-branching and increased side chain length are detrimental. Molecular models indicate that the mutants have impaired S2' binding sites and that beta-branching causes steric conflicts with the FXIa 140-loop, which could perturb the local tertiary structure of the protease domain. In conclusion, enzyme activity is impaired in FXIa when Gly193 is replaced by a non-Gly residue, and residues with side chains that branch at the beta-carbon have the greatest effect on catalysis and binding of substrates.
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PMID:Functional role of residue 193 (chymotrypsin numbering) in serine proteases: influence of side chain length and beta-branching on the catalytic activity of blood coagulation factor XIa. 1818 17

Heparin accelerates inhibition of factor XIa (fXIa) by the serpins antithrombin (AT) and C1-inhibitor (C1-INH) by more than 2 orders of magnitude. The mechanism of the heparin-mediated acceleration of fXIa inhibition by these serpins is incompletely understood, as heparin appears to interact with both the catalytic and noncatalytic domains of the protease. We replaced the basic residues of the fXIa 170 loop (Lys-170, Arg-171, Arg-173, Lys-175, and Lys-179; chymotrypsin numbering) with Ala, using an expression system that allows separation of the fXIa catalytic domain (CD) from noncatalytic domains. Heparin-mediated inhibition of 170 loop CD variants with AT was impaired 3-10-fold relative to that of the wild-type (CD-WT). In reactions with C1-INH, Arg-171 was the most critical residue contributing approximately 2-3-fold to heparin-mediated inhibition of CD-WT. A template mechanism did not fully account for the effect of heparin with either serpin, as the second-order inhibition rate constants did not exhibit a characteristic bell-shaped dependence on heparin concentration. Further studies revealed that the C1-INH inhibition of full-length fXIa containing Ala substitutions for basic residues of the 148 loop is not enhanced by heparin. Inhibition by AT of a full-length fXIa variant containing an Ala substitution for Arg-37 in the fXIa CD was approximately 5-fold greater than for wild-type fXIa in the absence of heparin. These results suggest that basic residues of the fXIa 170 loop form a heparin-binding site and that the accelerating effect of heparin on inhibition of fXIa by AT or C1-INH may be mediated by charge neutralization and/or allosteric mechanisms that overcome the repulsive inhibitory interactions of serpins with basic residues on the fXIa 148 and 37 loops.
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PMID:Characterization of a heparin-binding site on the catalytic domain of factor XIa: mechanism of heparin acceleration of factor XIa inhibition by the serpins antithrombin and C1-inhibitor. 1917 50


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