Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.4.21.4 (trypsin)
 42,187 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The properties of the functional groups in a protein can be used as built-in-probes of the structure of the protein. We have developed a general procedure whereby the ionization constant and chemical reactivity of solitary functional groups in proteins may be determined. The method may be applied to the side chain of histidine, tyrosine, lysine, and cysteine, as well as to the amino terminus of the protein. The method, which is an extension of the competitive labeling technique using [3H]- and [14C]1-fluoro-2,4-dinitrobenzene (N2ph-F) in a double-labeling procedure, is rapid and sensitive. Advantage is taken of the fact that after acid hydrolysis of a dinitrophenylated protein, a derivative is obtained which must be derived from a unique position in the protein. The method has been applied to the solitary histidine residue of lysozyme, alpha-lytic protease, and Streptomyces griseus (S.G.) trypsin, as well as to the amino terminus of the latter protein. The following parameters were obtained for reaction with N2ph-F at 20 degrees C in 0.1 N KCl: the histidine of hen egg-white lysozyme, pKa of 6.4 and second-order velocity constant of 0.188 M-1 min-1; the histidine of alpha-lytic protease, pKa of 6.5 and second-order velocity constant of 0.0235 M-1 min-1; the histidine of S.G. trypsin, pKa of 6.5 and second-order velocity constant of 0.0328 M-1 min-1; the valyl amino terminus of S.G. trypsin, pKa of 8.1 and second-order velocity constant of 0.403 M-1 min-1. In addition, the results obtained provide clues as to the microenvironments of these functional groups, and indicate that the proteins studied undergo pH-dependent conformational changes which affect the microenvironment, and hence the chemical reactivity of these groups.
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PMID:A competitive labeling method for the determination of the chemical properties of solitary functional groups in proteins. 0 42

1. The technique of differential thermal and proteolytic inactivation has been employed as a conformational probe for the lysine-sensitive aspartokinase (EC 2.7.2.4) of Escherichia coli B. 2. L-Amino acid inhibitors of this enzyme each induce a characteristic enzyme conformation. This is evidenced by rates of thermal and proteolytic inactivation and Arrhenius activation energies for thermal inactivation which are characteristic of the amino acid present. 3. Phenylalanine and leucine binding are mutually exclusive as evidenced by competitive behavior in thermal inactivation experiments, suggesting a hydrophobic amino acid binding site with broad specificity. 4. The phenylalanine-dependent conformation and the leucine-dependent conformation differ considerably. In comparison with the native enzyme, the former is more labile to proteolysis by trypsin whereas the latter is more stable. First-order rate constants for thermal inactivation of the phenylalanine- and leucine-dependent conformations are, respectively, about one-half and one-tenth that of the native enzyme. 5. Items 3 and 4 taken together suggest that the conformations are ligand induced and do not arise via ligand stabilization of spontaneously arising conformers.
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PMID:Conformations of lysine-sensitive aspartokinase. 0 8

The chemical modification of two new double-headed-protease inhibitors from black-eyed peas, a trypsin-chymotrypsin inhibitor (BEPCI) and a trypsin inhibitor (BEPTI) with dansyl chloride was investigated under various conditions. The NH2-terminal serine of both BEPCI and BEPTI, the 4 lysyl residues of BEPCI, and 4 of the 5 lysyl residues of BEPTI, could not be dansylated in the absence of urea. The single tyrosine per subunit of BEPCI and BEPTI was unreactive even in the presence of urea but could be labeled with half-site reactivity by the Celite method. Lysine, NH2-terminal serine, and tyrosine were reactive in fully reduced, carbamidomethylated BEPCI and BEPTI. Gel filtration was used to study the subunit interactions of BEPCI and BEPTI. At pH 8 or pH 3.0 there is a complex set of multiple equilibria with widely differing rates of attainment. We have found evidence for a rapid dimer-tetramer equilibrium, a distinct moderate rate dimer-tetramer equilibrium, a very slow monomer-dimer equilibrium, and postulate slow isomerization of the two forms of dimer and the two forms of tetramer. The monomer-dimer equilibrium is quite unusual in that the dimer is stabilized by chaotropic ions and even slightly by guanidine HC1. In contrast to the complex pattern seen in native BEPCI, the half-site, dansylated BEPCI exists at similar concentration exclusively as a tetramer at neutral pH.
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PMID:Double-headed protease inhibitors from black-eyed peas. III. Subunit interactions of the native and half-site chemically modified proteins. 0 94

The myeloma IgA protein produced by plasmacytoma XRPC-25, was isolated by affinity chromatography on dinitrophenyllysine-Sepharose. The affinity constant of the intact protein or its Fab' toward 2,4-dinitrophenyl-L-lysine (Dnp) was found to be 2.6 X 10(5) M-1. In order to prepare an Fv fragment (Hochman, J., Inbar, D., and Givol, D. (1973), Biochemistry 12, 1130) from this protein, the heavy and light chains were separated and the light chain was digested with trypsin at pH 8.2 to yield half a light chain. This digest was reassociated with the heavy chain and the recombinant was digested with papain at pH 5.7. Fractionation of this digest on a Sephadex G-75 column and Dnp-lysine-Sepharose resulted in the isolation of an Fv fragment which possesses one binding site for Dnplysine (Ka = 2.0 X 10(5) M-1). The active Fv fragment has a molecular weight of 23,400 and is composed of two peptide chains, each having a molecular weight of approximately 12,000. The N-terminal residues of these chains are aspartic and glutamic acids, which are also N-terminal in the heavy and light chains, indicating that the Fv is composed of VL and VH.
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PMID:Preparation of Fv fragment from the mouse myeloma XRPC-25 immunoglobulin possessing anti-dinitrophenyl activity. 0 96

Two thiol-activated endopeptidases with pH optima near pH 7.5 were isolated from the supernatant fraction of rabbit brain homogenates by DEAE-cellulose chromatography, gel filtration and isoelectrofocusing. Peptide bond hydrolysis was measured quantitatively by ion-exchange chromatography with an amino acid analyzer. Brain kininase A hydrolyzes the Phe5-Ser6 peptide bond in bradykinin (Bk), Arg1-Pro2-Pro3-Gly4-Phe5-Ser6-Pro7-Phe8-Arg9. It is isoelectric near pH 5.2 and has a molecular weight of approximately 71 000. The enzyme also hydrolyzes the Phe-Ser peptide bond in Lys-Bk, Met-Lys-Bk, des-Arg1-Bk, Lys9-Bk, Pro-Gly-Phe-Ser-Pro-Phe-Arg, and Gly-Pro-Phe-Ser-Pro-Phe-Arg, but does not hydrolyze (0.1%) this bond in des-Phe8-Arg9-Bk. Brain kininase B hydrolyzes the Pro7-Phe8 peptide bond in Bk. It is isoelectric at pH 4.9 and has a molecular weight of approximately 68 000. Brain kininase B also hydrolyzes the Pro-Phe bond in Lys-Bk, Met-Lys-Bk, Lys9-Bk, Ser-Pro-Phe-Arg, and Phe-Ser-Pro-Arg. Pretreatment of denatured kininogen with brain kininase A or B did not reduce the amount of trypsin-releasable Bk from this precursor protein, indicating that the Bk sequence, when part of a large protein, is not a substrate for either enzyme. However, kininase A and B hydrolyze the octadecapeptide Gly-Leu-Met-Lys-Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg-Ser-Val-Gin-Val. The data show that a large part of the C-terminal portion of bradykinin is important for the brain kininase A activity and, for both enzymes, the size of the peptide and presumably the residues adjacent to the scissle bond are important in determining the rate of peptide bond hydrolysis by these endopeptidases.
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PMID:Isolation of brain endopeptidases: influence of size and sequence of substrates structurally related to bradykinin. 0 20

Some properties of rat skin benzoylarginine-2-naphthylamide hydrolase types I (preparations I and AI) and II (preparations II and NII) were studied. Both types were activated by dithiothreitol and EDTA, but responded differently to 1 mM KCN, when benzoylarginine-2-naphthylamide (BANA) was used as a substrate: type I was inhibited, while type II was activated. When leucine-2-naphthylamide was used as a substrate, both types were activated by KCN. Thiol proteinase inhibiting substances, like heavy metals, iodoacetic acid, 4-chloromercuribenzoic acid, and tosyllysine chloromethylketone, inhibited the enzymes. Diisopropylfluorophosphate, phenylmethylsulfonyfluoride, 4-aminobenzamidine, and high-molecular-weight trypsin inhibitors were without effect. The substrate specificity of rat skin BANA hydrolase resembled that of an amino acid naphthylamidase, naphthylamides of methionine, lysine, arginine, and alanine being hydrolyzed most rapidly. The rate of hydrolysis of BANA was only 11% of that of methionine naphthylamide. Amino acid esters with a free alpha-amino group were also good substrates. The transformation of type II to type I at acidic pH was studied. During the transformation amino acids or peptides were formed and probably some inhibitor present in type II was destroyed proteolytically.
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PMID:Alpha-N-Benzoylarginine-2-naphthylamide hydrolase (cathepsin BI?) from rat skin. III. Substrate specificity, modifier characteristics, and transformation of the enzyme at acidic pH. 0 11

The active site of porcine enteropeptidase (EC 3.4.21.9) was investigated in order to characterize better both catalytic and binding sites. The participation of a serine and a histidine residue in the catalytic process was fully confirmed and the two residues were located on the light chain of the enzyme. The binding site was found to be composed of at least 2 subsites S1 and S2. The subsite S1 (similar to the trypsin-binding site) is responsible for the interactions with the small substrates of trypsin and the lysine side chain of trypsinogen, while subsite S2 (probably a cluster of lysines) is responsible for the interactions with the polyanionic sequence found in all trypsinogens. Binding of substrate by subsite S2 led to an increased efficiency of the catalytic site which can be correlated to the known high specificity of enteropeptidase.
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PMID:On the catalytic and binding sites of porcine enteropeptidase. 1 10

The reactive site peptide bond of the eggplant inhibitor against trypsin [EC 3.4.21.4] was identified by chemical modifications with 1,2-cyclohexanedione, 2,4,6-trinitrobenzenesulfonic acid, acetic anhydride and glyoxal, and by sequential treatments with trypsin and carboxypeptidase B [EC 3.4.12.3]. The inhibitor was significantly inactivated by chemical modifications of arginine residues, but was not affected by lysine modifications. Free arginine was released from the trypsin-modified inhibitor by carboxypeptidase B digestion, accompanied by a marked loss of inhibitory activity. A serine residue was newly exposed at the N-terminal amino acid of the inhibitor after modification with trypsin. The reactive site of the inhibitor against trypsin was concluded to be an arginylseryl bond. The inhibitor was completely inactivated by full reduction of its disulfide bonds.
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PMID:The reactive site of eggplant trypsin inhibitor. 1 22

A possible source of discrepancy between kinetic and spectroscopic studies of the active site ionizations in the enzyme trypsin (EC 3.4.21.4) could arise if a slow pH-dependent conformational change affected the rates at low pH. No such effect is observed within the time range of 1 min- 3 h when pre-incubation of trypsin at pH 2.0 or at pH 6.9 precedes the enzymatic hydrolysis of Nalpha-carbobenzoxy-L-lysine-p-nitrophenyl ester. The deacylation rate of this hydrolysis depends on a single pKa on the enzyme between pH 3 and pH 7.
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PMID:The effect of pre-incubation on trypsin kinetics at low pH. 1 15

A method of isolating preparations of pancreatic inhibitor of trypsin, bound with soluble polysaccharide carriers, is worked out. It is demonstrated that the reaction of a pancreatic inhibitor and cyanuric chloride-activated dextran proceeds for OH groups of tyrosine residues and for-epsilon-NH2 groups of lysine residues. A method is offered of the protection of amino groups with citraconic anhydride for the complete retaining of the inhibitory activity during attachment to dextran. Thermic denaturation of pancreatic inhibitor preparations at pH 4.7 and 97 degrees C is studied. It is found that the modification by 2-amino-4.6-dichloro-s-triazine stabilizes the protein molecule, while the interaction with the matrix of soluble dextran does not carry any contribution to thermostability of the pancreatic inhibitor.
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PMID:[Soluble high molecular weight derivatives of trypsin pancreatic inhibitor. Isolation and properties of dextran-bound pancreatic inhibitor]. 2 Jan 63


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