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 doughnut hypothesis of cytolysis by complement [Mayer, M. M. (1972) Proc. Nat. Acad. Sci. USA 69, 2954-2958] describes an annular structure made up of C5b-9 (complement factors C5b, C6, C7, C8, and C9) which becomes inserted in the lipid bilayer of the cell membrane, thus creating a hole. We now present initial explorations of this hypothesis. EAC1-6 and EAC1-7 (sheep erythrocytes carrying rabbit antibody and complement factors C1 through C6 or C1 through C7, respectively), prepared with either 125I-C3 or 125I-C5 were incubated with trypsin and the release of bound 125I was measured. In the case of 125I-C3, all of the radioactivity was released by trypsin from both intermediates. With 125I-C5, trypsin released all of the 125I from EAC1-6, but only 40-55% from EAC1-7. Possible reasons for resistance of the C5b subunit in EAC1-7 to tryptic digestion are discussed; in terms of the doughnut hypothesis it would be due to shielding by lipid molecules as a consequence of insertion into the lipid bilayer. In accord with this interpretation we have also found that C5b in EAC1-7, but not in EAC1-6, resists elution by 0.3 M NaC1. Similarly, we have found that 125I-C7 in EAC1-7 resists stripping by trypsin. Hence, we now propose the hypothesis that hydrophobic polypeptide chains from the C5b and the C7 subunits of C5b,6,7 complex become inserted in the phospholipid bilayer and that subsequent reactions with C8 and C9 open a channel across the membrane.
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PMID:On the mechanism of cytolysis by complement: evidence on insertion of C5b and C7 subunits of the C5b,6,7 complex into phospholipid bilayers of erythrocyte membranes. 106 Oct 92

The naturally occurring peptidyl protease inhibitor leupeptin (N-acetyl-L-leucyl-L-leucyl-L-argininal) has been prepared labeled with 13C at the argininal carbonyl. 13C chemical shift data for the trypsin-leupeptin inhibitor complex in the pH range 3.0-7.6 reveal the presence of two pH-dependent covalent complexes, suggestive of two interconverting diastereomers at the new asymmetric tetrahedral center created by covalent addition of Ser195 to either side of the 13C-enriched aldehyde of the inhibitor. At pH 7 two signals are observable at delta 98.8 and delta 97.2 (84:16 ratio), while at pH 3.0 the latter signal predominates. In the selective proton 13C-edited NOE spectrum of the major diastereomer at pH 7.4, a strong NOE is observed between the hemiacetal proton of the inhibitor and the C2 proton of His57 of the enzyme, thus defining the stereochemistry of the high pH complex to the S configuration in which the hemiacetal oxygen resides in the oxyanion hole. pH titration studies further indicate that the 13C chemical shift of the S diastereomer follows a titration curve with a pKa of 4.69, the magnitude of which is consistent with direct titration of the hemiacetal oxygen. Similar pH-dependent chemical shifts were obtained by using CPMAS 13C NMR, providing evidence for the existence of the same diastereomeric equilibrium in the solid state.
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PMID:Diastereotopic covalent binding of the natural inhibitor leupeptin to trypsin: detection of two interconverting hemiacetals by solution and solid-state NMR spectroscopy. 191 68

The crystal structure of subtilisin BPN' complexed with a proteinaceous inhibitor SSI (Streptomyces subtilisin inhibitor) was refined at 1.8 A resolution to an R-factor of 0.177 with a root-mean-square deviation from ideal bond lengths of 0.014 A. The work finally established that the SSI-subtilisin complex is a Michaelis complex with a distance between the O gamma of active Ser221 and the carbonyl carbon of the scissile peptide bond being an intermediate value between a covalent bond and a van der Waals' contact, 2.7 A. This feature, as well as the geometry of the catalytic triad and the oxyanion hole, is coincident with that found in other highly refined crystal structures of the complex of subtilisin Novo, subtilisin Carlsberg, bovine trypsin or Streptomyces griseus protease B with their proteinaceous inhibitors. The enzyme-inhibitor beta-sheet interaction is composed of two separate parts: that between the P1-P3 residues of SSI and the 125-127 chain segment (the "S1-3 site") of subtilisin and that between the P4-P6 residues of SSI and th 102-104 chain segment (the "S4-6 site") of subtilisin. The latter beta-interaction is unique to subtilisin. In contrast, the beta-sheet interaction previously found in the complex of subtilisin Novo and chymotrypsin inhibitor 2 or in the complex of subtilisin Carlsberg and Eglin C is distinct from the present complex in that the two types of beta-interactions are not separate. As for the flexibility of the molecules comprising the present complex, the following observations were made by comparing the B-factors for free and complexed SSI and comparing those for free and complexed subtilisin BPN'. The rigidification of the component molecules upon complex formation occurs in a very localized region: in SSI, the "primary" and "secondary" contact regions and the flanking region; in subtilisin BPN', the S1-3 and S4-6 sites and the flanking region.
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PMID:Refined crystal structure of the complex of subtilisin BPN' and Streptomyces subtilisin inhibitor at 1.8 A resolution. 192 Apr 11

The transition state of acylation step of trypsin catalysis was determined by molecular orbital calculations. The calculations were carried out at the RHF-LCAO-SCF approximation level with double zeta basis set (plus polarization functions). The role of His57 residue in the acylation step of the catalytic reaction of trypsin was analysed from a quantum mechanical point of view. The influences of surrounding residues, such as oxyanion hole and Asp102-, and the electrostatic effect of the other regions of the enzyme were also studied. His57 was proved to capture the proton from Ser195 side chain terminus with its lone pair and to transfer it to substrate with electrostatic assistance of Asp102- and oxyanion hole.
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PMID:Ab initio study on the transition state of acylation step of trypsin catalysis. 216 53

The precursor to the hole-forming toxin aerolysin has been purified in high yield from culture supernatants of a mutant of Aeromonas salmonicida containing the cloned structural gene. The mutant strain was generated by Tn5 mutagenesis. It released little or no protease or other extracellular proteins, including phospholipase, suggesting that it is a regulatory mutant. The absence of protease allowed the isolation of protoxin free from contaminating aerolysin. Typically, more than 50 mg of pure proaerolysin was obtained from 2 L of culture supernatant. The purified protein was completely unable to lyse human erythrocytes without prior activation with trypsin.
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PMID:Purification of cloned proaerolysin released by a low protease mutant of Aeromonas salmonicida. 219 Jun 17

Many unexpected biological functions as bioreactants of the intracellular proteases and their endogenous inhibitors have been found recently. Chymase and tryptase in histamine granules of mast cells and basophile cells play an important role in the process of IgE-mediated degranulation and in the formation of an allergic inflammation profile. Furthermore, the relationship between membrane proteases and their endogenous inhibitor has been taken up as a key and key-hole relation which plays an important role for special recognition apparatus of biological information like the relation of peptide hormones (growth factors) and their specific receptors. Amino acid sequences of the active site of trypstatin are homologous with the neutralizing epitope beta of gp120 of AIDS virus (HIV-1). The trypstatin and anti-tryptase M antibody inhibited syncytium formation in HIV infected Molt 4, clone 8 cells. Therefore, the relationship between tryptase M with trypstatin and the recognition site of epitope beta of HIV-1 with the receptor of helper T-cells are the common keys. The precursor of Alzheimer's deposition protein contains a Kunitz-type trypsin inhibitor domain. The A4-precursor proteins are located in axons of pyramidal neurons in brain and secretory granules of chromaffin cells in adrenal medulla. Those may be secreted into the extracellular milieu. We propose that the A4 inhibitor inhibits a special type of tryptase in the brain and disturbs the complete degradation of secreted A4-precursor protein causing amyloid deposition in alzheimer disease by abnormal proteolysis. Human c-Ha-ras p21 shows 58% homology with cystatin beta, an endogenous inhibitor of cathepsin. Actually, p21 inhibits cathepsin L specifically, but not cathepsin H, papain and cathepsin B. However, the metabolic significance of this inhibitory activity is still unknown.
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PMID:New biological functions of intracellular proteases and their endogenous inhibitors as bioreactants. 220 23

Factor IX is the zymogen of the serine protease factor IXa involved in blood coagulation. In addition to a catalytic domain homologous to the chymotrypsin family, it has Ca2+, phospholipid, and factor VIIIa binding regions needed for full biologic activity. We isolated a nonfunctional factor IX protein designated factor IXEagle Rock (IXER) from a patient with hemophilia B. The variant protein is indistinguishable from normal factor IX (IXN) in its migration on sodium dodecyl sulfate-gel electrophoresis, isoelectric point in urea, carbohydrate content and distribution, number of gamma-carboxyglutamic acid residues, and beta-OH aspartic acid content, and in its binding to an anti-IXN monoclonal antibody which has been shown previously to inhibit the interaction of factor VIIIa with factor IXaN. Further, IXER is cleaved to yield a factor IXa-like molecule by factor XIa/Ca2+ at a rate similar to that observed for IXN. However, in contrast to IXaN, IXaER does not bind to antithrombin-III (specific inhibitor of IXaN) and does not catalyze the activation of factor X (substrate) to factor Xa. To identify the mutation in IXER, all eight exons of IXN and IXER gene were amplified by the polymerase chain reaction technique and cloned. A single point mutation (G----T) which results in the replacement of Val for Gly363 in the catalytic domain of IXER was identified. Gly363 in factor IXa corresponds to the universally conserved Gly193 in the active site sequence of the chymotrypsin serine protease family. X-ray crystallographic data in the literature demonstrate a critical role of this Gly in stabilizing the active conformation of chymotrypsin/trypsin in two major ways: 1) in the formation of the substrate binding site; and 2) in the development of the oxyanion hole. Our computer structural data support a concept that the Gly363----Val change prevents the development of the active site conformation in factor IXa such that the substrate binding site and the oxyanion hole are not formed in the mutated enzyme.
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PMID:Experimental and theoretical evidence supporting the role of Gly363 in blood coagulation factor IXa (Gly193 in chymotrypsin) for proper activation of the proenzyme. 230 34

Aprotinin derivatives with decarboxylated lysine, arginine or valine at position 15, the P1 position of modified aprotinin, were produced semisynthetically. Modified aprotinin with oxidatively deaminated Arg1 and Ala16 was also synthesized. Specific reduction of this derivative yielded a modified aprotinin with lactic acid at position 16, the P'1 position. Only the aprotinin derivatives with decarboxylated Lys15 or Arg15 showed moderate inhibitory activity against trypsin and kallikrein, despite the absence of the carboxyl group. The KD values measured were in the range of 10(-7) M. The aprotinin derivative with decarboxylated valine showed no inhibitory activity; neither against trypsin, kallikrein and chymotrypsin, nor against the human leukocyte elastase. From these data it was concluded that the ion-pair interaction of the Lys15, or the Arg15 inhibitor side-chain with the aspartate in the trypsin specificity pocket is important for the inhibitory activity. Furthermore, the KD values indicated that the interaction of the reactive-site's carbonyl group with the enzyme's oxyanion hole also contributes to the inhibitory activity. These two interactions are important, but not essential for inhibitory activity. In contrast to these findings, the existence of an alpha-amino group at the P'1 position seems to be essential for inhibitory activity. The synthesized aprotinin derivatives lacking an alpha-amino group at this position were without any inhibitory activity against serine proteinases.
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PMID:Semisynthetic aprotinin derivatives with specific alterations at the reactive-site peptide bond can be used to study structure-function relationships. 247 74

The structure of the Asn102 mutant of trypsin was determined in order to distinguish whether the reduced activity of the mutant at neutral pH results from an altered active site conformation or from an inability to stabilize a positive charge on the active site histidine. The active site structure of the Asn102 mutant of trypsin is identical to the native enzyme with respect to the specificity pocket, the oxyanion hole, and the orientation of the nucleophilic serine. The observed decrease in rate results from the loss of nucleophilicity of the active site serine. This decreased nucleophilicity may result from stabilization of a His57 tautomer that is unable to accept the serine hydroxyl proton.
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PMID:The three-dimensional structure of Asn102 mutant of trypsin: role of Asp102 in serine protease catalysis. 311 42

The hole-forming toxin aerolysin was shown to aggregate after binding to erythrocytes at 37 degrees C. Although the protein also bound and aggregated at 4 degrees C, hole formation was not observed, indicating that aggregation preceded penetration of the lipid bilayer. Aggregation, but not binding, could be blocked by pretreatment of the toxin with diethyl pyrocarbonate, a histidine-reactive reagent. This resulted in inactivation of the toxin. Incubation of aerolysin with glycophorin purified from human erythrocytes caused aggregation and complete inactivation. Erythrocytes which lacked glycophorin were less sensitive to the toxin. Proaerolysin, the inactive precursor of aerolysin, also bound to erythrocytes; however, it did not aggregate, nor did it aggregate when preincubated with glycophorin. The protoxin could be activated by treatment with trypsin even after it had bound to erythrocytes. Activation could also be achieved by reaction of proaerolysin with a variety of other proteases, each of which brought about a similar reduction in protein molecular weight. The activated protein was resistant to further proteolysis. These results indicate that aggregation is a necessary step in hole formation and that the sites on aerolysin required for binding and for aggregation and hole formation are separate.
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PMID:The cytolytic toxin aerolysin must aggregate to disrupt erythrocytes, and aggregation is stimulated by human glycophorin. 328 5


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