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:3.4.21.1 (chymotrypsin)
10,938 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The reactive-site sequence of a proteinase inhibitor can be written as . . . -P3-P2-P1-P'1-P'2-P'3- . . . , where-P1-P'1-denotes the reactive site. Three semisynthetic homologues have been synthesized of the bovine trypsin-kallikrein inhibitor (Kunitz) with either arginine, phenylalanine or tryptophan in place of the reactive-site residue P1, lysine-15. These homologues correspond to gene products after mutation of the lysine 15 DNA codon to an arginine, phenylalanine or tryptophan DNA codon. Starting from native (virgin) inhibitor, reactive-site hydrolyzed, still active (modified) inhibitor was prepared by chemical and enzymic reactions. Modified inhibitor was then converted into inactive des-Lys15-inhibitor by reaction with carboxypeptidase B. Inactive des-Lys15-inhibitor was reactivated by enzymic replacement of the P1 residue according to Leary and Laskowski, Jr. The introduction of arginine was catalyzed by an inverse reaction with carboxypeptidase B, while phenylalanine or tryptophan were replaced by carboxypeptidase A. The reactivated semisynthetic inhibitors were trapped by complex formation with either trypsin or chymotrypsin. The enzyme - inhibitor complexes were subjected to kinetic-control dissociation, and the semisynthetic virgin inhibitors were isolated. The inhibitory properties of the semisynthetic inhibitors have been investigated against bovine trypsin and chymotrypsin and against porcine pancreatic kallikrein and plasmin. The homologues with either lysine or arginine in the P1 position are equally good inhibitors of trypsin, plasmin and kallikrein. The Arg-15-homologue is a slightly more effective kallikrein inhibitor than the Lys15-inhibitor. The semisynthetic phenylalanine and tryptophan homologues, however, are weak inhibitors of trypsin and still weaker inhibitors of kallikrein, but are excellent inhibitors of chymotrypsin. Their association constant with chymotrypsin is at least ten times higher than that of native Lys-15-inhibitor. A dramatic specificity change is observed with the phenylalanine and tryptophan homologues, which in contrast to the native inhibitor do not at all inhibit porcine plasmin. Thus, the nature of the P1 residue strongly influences the primary inhibitory specificity of the bovine inhibitor (Kunitz).
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PMID:Replacement of lysine by arginine, phenylalanine and tryptophan in the reactive site of the bovine trypsin-kallikrein inhibitor (Kunitz) and change of the inhibitory properties. 12 27

The exposure of apolipoproteins at the surface of human plasma high density lipoproteins (HDL) was assessed by their accessibility to agarose-immobilized forms of trypsin and chymotrypsin. Proteolysis of lipid-free apolipoproteins and the lipoprotein subfractions HDL2 (d = 1.08--1.125 g/ml) and HDL3 (d = 1.125--1.195 g/ml) that differ in lipid-to-protein ratio was compared by polyacrylamide gel electrophoresis and isoelectric focusing of the apolipoproteins and peptide fragments and by quantitation of the various carboxyl-terminal groups formed. Gel filtration of the proteolyzed lipoproteins on Sephadex G-150 column indicated that more than 90% of the apolipoproteins and peptides remain associated with lipoprotein complexes. Proteolysis of lipoproteins occurred more slowly and with less fragmentation of the lipoproteins and apolipoproteins than proteolysis of thelipid-free apolipoproteins or the proteolysis of lipoproteins by soluble proteases reported by other investigators. The difference in lipid content of HDL2 and HDL3 made little difference in their proteolysis. Proteolysis of the lipoproteins by agarose-trypsin was more rapid at 37 degrees C than at 22 degrees C, but the proteolytic products were similar and differed from the products from the lipid free proteins. Peptide fragments from lipoproteins were larger than those from lipid-free proteins, which suggests masking of potentially cleavable groups by lipid. The amounts (mol/g protein) of new carboxyl-terminal tyrosine and phenylalanine released by agarose -chymotrypsin were much greater from the lipid-free proteins, but about 3/4 of the tryptophan residues were inacessible in both lipoproteins and lipid-free proteins. In agarose-trypsin digestion, lysine residues were slightly more masked than arginine in the absence of lipids and much more so in the lipoproteins. However, in the lipoproteins apoA-II, which contains lysine but no arginine, was cleaved more rapidly and extensively by agarose-trypsin than apoA-I.
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PMID:Surface exposure of apolipoproteins in high density lipoproteins. I. Reactivities with agarose-immobilized proteases. 20 44

Chymotrypsinogen A and alpha-chymotrypsin are both nitrated at tyrosines 146 and 171 by reaction with tetranitromethane. This substitution was essentially without influence on the overall rate constant for hydrolyses of N-acetyl-L-tryptophan methyl ester and N-acetyl-L-tyrosine ethyl ester catalyzed by alpha-chymotrypsin and delta-chymotrypsin, prepared by fast tryptic activation of nitrated chymotrypsinogen. With both ester substrates Km was doubled for nitrated alpha-chymotrypsin. Nitrated alpha-chymotrypsin, nitrated delta-chymotrypsin and delta-chymotrypsin could all bind N-acetyl-L-tryptophan methyl ester at alkaline pH, in contrast to alpha-chymotrypsin. The dissociation constant, Kd, of the complex of alpha-chymotrypsin and basic pancreatic trypsin inhibitor was lowered ten-fold relative to the constant obtained with unmodified alpha-chymotrypsin. The nitrated delta-chymotrypsin and delta-chymotrypsin showed identical Kd values. The nitrated alpha-chymotrypsin is inactivated faster at pH 8.0 and 8.5 than alpha-chymotrypsin and apparently by a different mechanism.
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PMID:Enzymic properties of nitrated alpha-chymotrypsin and delta-chymotrypsin. 24 45

A recent paper [Chibber, B. A. K., Tomich, J. M., Mertz, E. T. & Viswanatha, T. (1977) Proc. Natl. Acad. Sci. USA 74, 510-514] presented evidence that was taken to support the existence of an intermediate in the deacetylation of acetylchymotrypsin. It was observed that deacylation, as measured by following the decrease in [(14)C]acetylchymotrypsin (decrease in acid-precipitable radioactivity), occurred at 1/10 the rate of reactivation, as measured by return of activity toward N-acetyl-L-tyrosine ethyl ester. Our experiments have shown that, at pH 6, the deacylation rate constant (measured by the loss of [(14)C]acetylchymotrypsin and by the formation of [(14)C]acetate) is identical (within experimental error) with the rate constant for reactivation (measured by determining the activity of aliquots of reactivating enzyme against N-acetyl-L-tryptophan ethyl ester) and with K(cat) for the turnover of p-nitrophenyl acetate by alpha-chymotrypsin. Part of the 10-fold greater reactivation rate observed by Chibber et al. has been shown to be due to the presence of 10% (vol/vol) isopropanol in their reactivation mixture, and it is argued that the balance of the effect is a manifestation of the "indole effect" produced by the simultaneous presence of 10 mM N-acetyl-L-tyrosine ethyl ester throughout the reactivation experiments. The results presented are entirely consistent with the three-step mechanism of catalysis by alpha-chymotrypsin and negate the existence of the proposed additional acetyl-enzyme intermediate.
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PMID:Absence of evidence for an intermediate in the deacetylation of acetylchymotrypsin. 27 31

The chymotrypsin-like proteins (chymotrypsin-CT,chymotrypsinogen-CTG, trypsin-T and modified chymotrypsins-at Met 192-MCT and at Tyr 146, 171-TCT), gamma-irradiated in the presence of air, were investigated. Irradiation leads to the unfolding of the native structure of CT-like proteins both in solution and in the dry state, which was shown by the tryptophan fluorescence, viscosimetry and microcalorimetry. The radiation yield of unfolded molecules Gconf was estimated and compared with (1) the rate constants for the reactions of OH-radicals with the proteins as determined by the p-nitrosodimethylaniline, (2) general stability of protein globule using the difference of the energies of the unfolded and globular conformations and (3) the radiation yield of tryptophan destruction in proteins-G-trp. There was a correlation between the values of Gconf and G-trp. The ratio G-trp/Gconf, which defines the number of destroyed tryptophan residues for one unfolded protein molecule, was constant within the limits of error. For CT, MCT, TCT and CTG, this ratio was on the average 3-2, and for T it was 2-2 residues. These facts point to the role of tryptophan destruction in the unfolding of the native structure of CT-like proteins on irradiation.
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PMID:Gamma-irradiated chymotrypsin-like proteins. I. Structural changes. 30 Jul 18

The quenching of tryptophan fluorescence by N-bromosuccinamide, studied by the fluorescence stopped-flow technique, was used to compare the reactivities of tryptophan residues in protein molecules. The reaction of N-bromosuccinamide with the indole group of N-acetyltryptophanamide, a model compound for bound tryptophan, followed second-order kinetics with a rate constant of (7.8 +/- 0.8) . 10(5) dm3 . mol-1 . s-1 at 23 degrees C. The rate does not depend on ionic strength or on the pH near neutrality. The non-fluorescent intermediate formed from N-acetyltryptophanamide on the reaction with N-bromosuccinamide appears to be a bromohydrin compound. The second-order rate constant for fluorescence quenching of tryptophan in Gly-Trp-Gly by N-bromosuccinamide was very similar, (8.8 +/- 0.8) . 10(5) dm3 . mol-1 . s-1. Apocytochrome c has the conformation of a random coil with the single tryptophan largely exposed to the solvent. The rate constant for the fluorescence quenching of the tryptophan in apocytochrome c by N-bromosuccinamide was (3.7 +/- 0.3) . 10(5) dm3 . mol-1 . s-1. The fluorescence quenching by N-bromosuccinamide of the tryptophan residues incorporated in alpha-chymotrypsin at pH 7.0 showed three exponential terms from which the following rate constants were derived: 1.74 . 10(5), 0.56 . 10(5) and 0.11 . 10(5) dm3 . mol-1 . s-1. This protein is known to have eight tryptophan residues in the native state, six residues at the surface, and two buried. Three of the surface tryptophans have the indole rings protruding out of the molecule and may account for the fastest kinetic phase of the quenching process. The intermediate phase may be due to three surface tryptophans whose indole rings point inwards, and the slowest to the two interior tryptophan residues.
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PMID:The reactivity of tryptophan residues in proteins. Stopped-flow kinetics of fluorescence quenching. 45 50

The interaction between BPN' or Carlsberg subtilisins and peptides of the type Ac-Glyn-X-OMe (n = 0, 1, 2, 3), where X denotes one of five different aromatic amino acids, was investigated to elucidate the effect of the secondary interaction on catalysis in relation to the nature of the X residue. The increase in interaction upon elongation of the chain was accompanied by a large increase in kcat but with no marked change in Km in all the series of sensitive substrates. The peptides containing 2-(2-nitro-4-carboxyphenylsulfenyl)-tryptophan, however, acted as competitive inhibitors and exhibited an invariant dissociation constant in spite of the different chain lengths. These observations suggest that the secondary enzyme-substrate interaction induces a conformational change in the active site of the enzyme or in the substrate in such a way as to lower the activation energy and to form a stabilized transient complex. In this respect, BPN' and Carlsberg subtilisins are similar to porcine pepsin and Streptomyces griseus protease 1 rather than to alpha-chymotrypsin.
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PMID:Interactions of BPN' and Carlsberg subtilisins with peptides containing aromatic amino acids at the C-terminus. Specific rate enhancement due to the secondary enzyme-substrate interaction. 47 43

Though DNase does not contain any cysteine residues, incubation of the enzyme with 2-nitro-5-thiocyanobenzoic acid in the presence of Ca2+ at pH values above 7.5 results in an irreversible inactivation of the enzyme. The inactivation also occurs when Ca2+ is replaced by Mg2+, but not in their absence. Amino acid analyses after acid hydrolyses of the completely inactivated ant the native enzymes show no significant differences in composition, including tryptophan and half-cystine residues. However, sodium dodecyl sulfate gel electrophoresis indicates enzyme cleavage by the treatment with 2-nitro-5-thiocyanobenzoic acid. This reagent does not inactivate chymotrypsin and lysozyme, and under conditions where bovine DNase is inactivated, does not inactivate other nucleases such as ribonuclease, snake venom phosphodiesterase, and spleen acid DNase. However, it inactivates malt DNase and can, therefore, be considered a specific inhibitor of DNase I. The inactivation kinetics is pseudo-first order, resembling Michaelis-Menten, with an affinity constant of 16.7 mM. It is the cyano group, not the thionitrobenzoic acid of 2-nitro-5-thiocyanobenzoic acid that reacts to form cyano-DNase.
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PMID:Inactivation of bovine pancreatic DNase by 2-nitro-5-thiocyanobenzoic acid. I. A novel inhibitor for DNase I. 48 54

The binding of N-acetyl-tryptophan to the monomeric and dimeric forms of alpha-chymotrypsin in I = 0.2 acetate-chloride buffer, pH 3.86, has been studied quantitatively. Equilibrium sedimentation studies in the absence of inhibitor yielded a dimerization constant of 3.5 L/g. This value was confirmed by frontal gel chromatography of the enzyme on Bio-Gel P-30, which was also used to establish that N-acetyl-L-tryptophan binds preferentially to monomeric enzyme. From kinetic studies of competitive inhibition with N-acetyl-L-tryptophan ethyl ester as substrate, an equilibrium constant of 1300 M-1 was determined for the binding of N-acetyl-L-tryptophan to monomeric alpha-chymotrypsin. An intrinsic binding constant of 250 M-1 for the corresponding interaction with dimeric enzyme was calculated on the basis of these results and binding data obtained with concentrated (18.5 g/L) alpha-chymotrypsin. The present results refute earlier claims for exclusive binding of competitive inhibitors to monomer and also those for equivalence of inhibitor binding to monomeric and dimeric forms of alpha-chymotrypsin.
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PMID:Evaluation of equilibrium constants for the binding of N-acetyl-L-tryptophan to monomeric and dimeric forms of alpha-chymotrypsin. 51 37

The largest fragment produced by complete cyanogen bromide digestion of the alpha chain of human fibrinogen contains 236 residues and has a calculated molecular weight of 23,949. The complete amino acid sequence of the fragment was determined by the isolation of peptides generated by plasmin, trypsin (including digestion of citraconylated material), staphylococcal protease, and chymotrypsin. In addition, some key subfragmentation was achieved by selective chemical cleavage at tryptophan residues. The fragment has an unusual amino acid composition, more than half of its residues being glycine, serine, threonine, and proline. There are very few nonpolar residues, although 7 of the alpha-chain's 10 tryptophans occur in this fragment. The fragment contains 2 cysteine residues located 30 residues apart which are connected by an intrachain disulfide bond in the native molecule. The tryptophans occur with a definite periodicity that highlights a series of 13-residue homology repeats. The fragment also contains the two principal alpha-chain cross-linking sites.
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PMID:Amino acid sequence studies on the alpha chain of human fibrinogen. Complete sequence of the largest cyanogen bromide fragment. 51 44


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