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 reaction between chymotrypsin and N-acetyl-L-phenylalanine p-nitroanilide has been studied at subzero temperatures in fluid aqueous dimethyl sulfoxide solvent. Following initiation of the reaction at temperatures as low as -90 degrees C, a series of four reactions prior to the normal rate-limiting step (acylation) was detected spectrophotometrically. Various experimental observations have led to the following interpretation of these reactions. Reaction 1 corresponds to the binding of substrate yielding the initial Michaelis complex. Reactions 2 and 3 are two pH-independent reactions, ascribed to substrate-induced changes in the positions of active-site groups. Reaction 4 is a pH-dependent reaction (pK = 5.9) which involves the imidazole of His-57 but which is not the formation of a tetrahedral intermediate, oxazolinone, or acyl enzyme. The slowest detected step corresponded to the acylation reaction. No evidence for the accumulation of a tetrahedral intermediate was obtained. Spectral, kinetic, and thermodynamic data for these reactions are presented, as is justification for the relevance of these findings to the reaction under physiological conditions. These results demonstrate the utility of subzero temperatures in enzyme mechanism studies, especially with regard to allowing the accumulation of intermediates which may be quite stable at appropriate values of pH and low temperature.
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PMID:Cryoenzymology of chymotrypsin: the detection of intermediates in the catalysis of a specific anilide substrate. 94 50

Effects of hydrogen-bonding interactions of amide groups on reactivity of esters to alpha-chymotrypsin were studied. Of the methyl esters studied, only that from acetyl-L-phenylalanine has k3 rate-limiting. In methyl beta-phenylpropionates an alpha-acetamido substituent increased k2 greater than 550 times, k3 approximately 5 times; an alpha-acetylclycyloxy substituent increased k2 approximately 2 times, k3 approximately 6 times, both in comparison with the alpha-acetoxy esters. Essentially all carboxamidomethyl esters studied have k3 rate-limiting; reactivity to hydroxide is only 4 times that of methyl esters. In alpha-substituted beta-phenylpropionates, carboxamido-methyl esters show k2 values greater than 110 times greater than 280 times, greater than 26 times, and 7 times the k2 values of the methyl esters for the alpha substituents, acetoxy, acetylglycyloxy, hydroxy, and hydrogen, respectively. In esters of alpha-acetamido acids, carboxamidomethyl esters show k2 values 44 times, greater than 110 times, greater than 12 times, and approximately 33 times the k2 values of the methyl esters of glycine, alanine, leucine, and phenylalanine, respectively. Cyanomethyl esters also had k3 rate-limiting. Hydrogen-bonding to the enzyme of either an alpha-acetamido group or a carboxamidomethyl group combined with bonding of the beta-aryl group, orients the hydrolyzing groups properly, increasing k2. Hydrogen-bonding of both alpha-acetamido and carboxamido-methyl groups is effective to a lesser degree. The amide group appears to have small effects on Ks as hydrogen bonding is balanced by desolvation. It is proposed that desolvation during bonding increases k2 and Ks.
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PMID:Carboxamidomethyl esters as reactive substrates for alpha-chymotrypsin. Orientational effects of hydrogen-bonding interactions. 94 6

The 5-dimethylaminonaphthalene-1-sulfonyl group was specifically introduced into the active site region of the serine proteinases: alpha-chymotrypsin, trypsin and subtilisin Carlsberg by the method of affinity-labeling. The resulting fluorescent derivatives were studied by a variety of fluorescence techniques and the results were correlated with structural data available on these enzymes from X-ray analysis. As model compounds for the Dns-proteinases, the absorption and fluorescence properties of Dns-amide and Dns-ethyl ester were studied in ethanol/water and p-dioxane/water mixtures. The fluorescence emission transtion energies and quantum yields were related to four commonly employed solvent-polarity scales. Best correlations for different solvents were obatined with the empirical "Z" and "Y" scales. From inspection of the fluorescence emission transition energies of the Dns group in the Dns-proteinases and comparision with the model compound studies it was possible to assign "Z" values for the apparent microenvironment polarities of the Dns group in the Dns-proteinases. The apparent polarities of the microenvironments of the Dns group in Dns-Ser 195-chymotrypsin (Dns-chymotrypsin (I)); (Dns-Phe-CH2)-His 57-chymotrypsin; (Dns-Lys-CH2)-His 46-trypsin; and Dns-Ser 221--subtilisin Carlsberg (Dns-subtilisin (I)) are in the range of 89.5-92.5 on the "Z" scale. The apparent microenvironment polarity of the Dns group in Dns-Ser 183-trypsin (Dns-trypsin (I)) appears to be below 76.7 on the "Z" scale. The Dns group in Dns-chymotrypsin (I) and (Dns-Phe-CH2)-His 57-chymotrypsin appears to be rigidly bound as evaluated by fluorescence polarization studies. The effect of 2H2O on the fluorescence emission quantum yields of Dns-amide and Dns-ethyl ester was examined. In both cases the ratios of quantum yields in 2H2O:ethanol (8:2) to quantum yields in H2O:ethanol (8:2) was about 1.8. The 2H2O effect upon the fluorescence emission quantum yields of the Dns group has been used to investigate solvent accessibility of this chromophore in the Dns-proteinases. Acessibility studies using 2H2O are very promising and have some definite advantages over other existing methods. Energy transfer between the Trp residues and the bound Dns group was investigated in the Dns-proteinases. The mean transfer distance calculated from the observed transfer efficiencies are 18.1 A, 19.7 A and 18.4 A for (Dns-Phe-CH2)-His 57-chymotrypsin, Dns-chymotrypsin (I) and (Dns-Lys-CH2)-His 46-trypsin, respectivly. From models built using X-ray crystallographic coordinates for the protein atoms, the mean distance of separation between the Trp residues and the bound Dns group for the same set of conjugates ar 18.6 A, 17.5 A and 17.5 A, respectively. Considering the inherent difficulties in energy transfer studies, the results are in excellent agreement with the X-ray data.
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PMID:Specific fluorescent derivatives of macromolecules. A fluorescence study of some specifically modified derivatives of chymotrypsin, trypsin and subtilisin. 95 53

The specific cyanate reagentN-acetyl-p-cyanato-L-phenylalanine ethyl ester (compound 1) was synthesized in an attempt to selectively modify the binding pocket of chymotrypsin (EC 3.4.21.1) while leaving the catalytic residues un touched. The reagent reacts with chymotrypsin to yield chiefly an inactive derivative 3a, with the active site Ser-195 carbamylated and the alpha-amino group, with the reagent 1 yields a modified enzyme(compound 4) with an additional carbamyl group on Ser-195.Neither derivative 3a nor 4reacts with diisopropylfluorophosphate under conditions where chymotrypsinogen is modified, indicating that Ser-195 is altered. Both derivatives 3a and 4 are retained on a 4-phenylbutylamine affinity colomn demonstrating that the substrate binding pocket is intact in both derivatives. The results indicate the potential value of aryl cyanates as protein reagents for the selective modification of nucleophilic sites. However, it is apparent that reaction at unreactive residues in the binding pocket of chymotrypsin with cyanates or similar reagents will require blockage of the more nucleophilic catalytic residues.
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PMID:Modification of chymotrypsin with the specific cyanate reagent N-acetyl-p-cyanato-l-phenylalanine ethyl ester. 95 38

Activities of hydrolytic enzymes on the surface of monkey kidney, canine kidney, L. FM3A and various tumor cells were determined and compared with those in the cell homogenate. Although aminopeptidase (EC 3.4.11.-) activities were always detected on the surface membrane in mammalian cells, trypsin, chymotrypsin and elastase activities were not detected while slight glycosidase activity was detected in a suspension of cultured cells. The activities of alanine-, leucine-, methionine- and phenylalanine-aminopeptidases were rather high but aminopeptidase A, proline-, valine-, glycyl propline dipeptidyl-and glycyl propyl leucine-tripeptidyl-aminopeptidases showed relatively low activities. Aminopeptidase activity was also demonstrated in the isolated membrane fractions. The specific activities of enzymes in these membrane fractions were not significantly greater than in cell homogenate so it was concluded that these enzyme activities were rather loosely bound to the cell membrane. Further evidence for the localization of the aminopeptidase activities on the cell surface was obtained by using glass-bead-bound substrate and detecting the release of the terminal residues. When bestatin, a specific inhibitor against aminopeptidase B and leucine aminopeptidase, was included in the assay system for the enzyme activities on the cell surface, the enzymes were commonly inhibited in all types of cells.
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PMID:Aminopeptidase activities on the surface of mammalian cells. 99 Mar 9

Bovine immunoglobulins (IgG1 type) have been isolated from colostral whey. Hydrolysis by pronase, trypsin and (or) chymotrypsin yield several glycopeptides structural studies of which lead to the following results. 1. IgG1 colostral immunoglobulins possess two glycan moieties which are linked to the peptidic chain by an N-(beta-aspartyl)-N-acetylglucosaminylamine bound. 2. The peptidic sequence around the linkage region has been determined by classical methods and is as follows: Thr-Lys-Pro-Arg-Glu-Glu-Gln-Phe-Asn(Glycan)-Ser-Thr-Tyr-Arg. 3. The following procedures: partial acidic hydrolysis, periodic oxidation, hydrazinolysis-nitrous deamination, methylation and use of specific glycosidases allowed us to determine the structure of the glycan moieties which fit with the general following scheme: (see article) Thus they could be related to the general glycan structure so-called of "N-acetyllactosamine type" because they possess the pentasaccharidic core common to numerous glycoproteins Man alpha 1 leads to [Man alpha 1 leads to 6] Man beta 1 leads to 4 GlcNAc beta 1 leads to 4 GlcNAc beta 1 leads to Asn on which are conjugated 2 N-acetyllactosamine residues. Besides they present a microheterogeneity which is due to the varying number of additional N-acetylneuraminic acid and fucose residues. 4. These structures are compared to various immunoglobulin structures proposed by others: bovine serum IgG and human serum IgG, IgE and IgA.
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PMID:[Complete structure of glycopeptides isolated from IgG immunoglobulins of cow colostrum]. 99 Mar 35

Fourteen 3-carboxypropionyl-tripeptide-p-nitroanilides of the general formula 3-carboxypropionyl-alanyl-alanyl-Y-p-nitroanilide (Y = glycine, norvaline, S-methylcysteine, valine, norleucine, S-ethylcysteine, methionine, leucine, isoleucine, phenylalanine, tyrosine, S-benzylcysteine, Calpha-phenylglycine, and proline) were synthesized and their cleavage by elastase, trypsin, and chymotrypsin (Km, kcat and kcat/Km) was determined. The significance of amino acid residues in the position of Y was evaluated firstly with respect to their structure (topographically), and secondly with respect to their free energy (thermodynamically). The alanine residue substrate was cleaved best by elastase, the phenylalanine substrate by chymotrypsin. Trypsin cleaved two substrates only, that is those containing a phenylalanine and a tyrosine residue. The optimum length of the elastolytic substrates was studied in a series of N-3-carboxypropionyl-(Ala)n-p-nitroanilides (n = 1, 2, 3, 4, 5), N-3-carboxypropionyl-(Gly)n-p-nitroanilides (n = 1, 2, 3), and in p-nitroanilides of fatty acids with two to seven carbon atoms. Elastase cleaved tri, tetra, and pentapeptides of alanine. p-Nitroanilides of the glycine series, as well as p-nitroanilides of fatty acids were not cleaved. 3-Carboxypropionyl-tetra-alanine-p-nitroanilide was the most suitable substrate so far found for elastase cleavage; it is not cleaved by trypsin nor chymotrypsin. The optimum distance between Y and the terminal anionic carboxyl residue was found to be 1.8 nm in elastolytic substrates.
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PMID:p-Nitroanilides of 3-carboxypropionyl-peptides. Their cleavage by elastase, trypsin, and chymotrypsin. 99 49

Characterization of the cyanogen bromide (CNBr) fragments of the beta chain of human haptoglobin revealed five major fragments resulting from cleavage of four methionyl residues. The fragments were isolated by gel filtration in guanidine-HCl on Sepharose 6B and Bio-Gel P10 and P60. Compositional analyses of the five cyanogen bromide fragments accounted for 248-253 amino acid residues in agreement with the number of residues determined for the intact beta chain. Most of the carbohydrate was attached to CNBr II. Automated amino-terminal sequence analysis and carboxyl-terminal hydrolysis with carboxypeptidase of the haptoglobin beta chain and cyanogen bromide fragments identified 139 residues, or about 55% of the beta-chain molecule. The placement of the fragments within the beta-chain molecule was established by sequence analysis of whole beta chain and a plasmin cleavage fragment. The position of CNBr V was confirmed by the absence of homoserine or homoserine lactone. Cyanogen bromide reaction of intact haptoglobin 1-1 resulted in the isolation of a beta-chain fragment, CNBr III, covalently attached to the intact alpha1 chain by a single disulfide bond. The beta chain was shown to have primary structural similarities to the chymotrypsin family of serin eproteases. Partial sequence analysis of CNBr V established the region which is comparable to the serine-195 active-site region: /Asp-Thr-Cys-Tyr-Gly-Asp-Ala-Gly-Ser-Ala-Phe/ (residues 189-199, chymotrypsinogen A numbering). The active-site serine-195 is replaced by alanine; however, the specificity residue of the trypsin-like enzymes, Asp-189, is preserved. Several minor cyanogen bromide cleavage products were also identified in yields of up to 15%. These minor cleavage products give evidence that tryptophanyl residues in proteins, or glycoproteins, are also susceptible to cyanogen bromide cleavage.
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PMID:Characterization of the cyanogen bromide fragments of the beta chain of human haptoglobin. 99 9

Affinity-labeling reagents are useful for the inactivation of proteases in vivo but are apparently limited in application by the possibility of side reactions. In addition, increased specificity would be desirable. Substrate-derived chloromethyl ketones, one class of protease inhibitors, have been examined with the hope that replacement of the departing group halogen by other substituents might lead to improved inhibitor characteristics. Analogues of Z-Phe-Ch2X were synthesized in which X is a sulfonate or carboxylate substituent and examined as inactivators of chymotrypsin. The sulfonate esters were found to be more reactive than the previously studied halogen derivatives.
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PMID:Active-site-directed alkylation of chymotrypsin by reagents utilizing various departing groups. 100 4

The sequences of amino acid residues at the amino and carboxyl terminus and around the reactive sites of the trypsin chymotrypsin inhibitor PCI 3 from the seeds of runner beans (Phaseolus coccineus L.) were estimated by aminopeptidase O and carbosypeptidase A degradation before and after enzymatical modification with trypsin or chymotrypsin. Beginning at the amino terminus the sequences are :Ser-Glu-Ala-Gly-Gln-...,...-Ile-Tyr-Lys-Ser-Gln-(Pro)-...with Lys-Ser as reactive site against trypsin, ...-Asp-Val-Ala-Leu-Ser-(Pro)-...with Leu-Ser as reactive site against alpha-chymotrypsin, and ...-Thr-Arg-Ala-Lys-Phe-Leu as C-terminus. The importance of the serine residue in the reactive sites concerning the specificity of inhibitors is discussed.
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PMID:[Trypsin and chymotrypsin inhibitors in leguminosae VII. Partial amino acid sequences of the trypsin chymotrypsin inhibitor PCI 3 from Phaseolus coccineus (author's transl)]. 100 24


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