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 kinetics of the acylation of alpha-chymotrypsin by a series of substituted phenyl p-nitrobenzoates have been studied by stopped flow and conventional spectrophotometry. Electron withdrawal in the leaving group accelerates the rate of acylation, and the p value obtained for eight esters is +1.96. The pH- and pD-independent acylation rate constants are, respectively, 1.40 X 10(4) M-1S-1 and 1.23 X 10(4) M-1S-1 for p-nitrophenyl p-nitrobenzoate, and, respectively, 2.19 X 10(3) M-1S-1 and 1968 X 10(3) M-1S-1 for p-nitrophenyl benzoate at 25 degrees. An analysis of structure-reactivity results and kinetic solvent isotope effects indicates a mechanism for acylation by phenylbenzoates in which initial reaction is a nucleophilic attack by an imidazole of the enzyme (His 57). Subsequently, there is rapid transfer of the acylating group to the serine 195 from the acylimidazole species. The kinetic solvent isotope effects for acylation by p-nitrophenyl phenyl acetate and p-nitrophenyl phenyl acetate and p-nitrophenyl hydrocinnamate, in 5%, v/v, acetonitrile, are 1.3 and 2.0, respectively. The latter ester is inhibited more than is p-nitrophenyl benzoate when 5%, v/v, dioxane is substituted for 5%, v/v, acetonitrile as co-solvent. In the presence of 5%, v/v, dioxane a change in the kinetic solvent isotope effect to 1.7 is found for p-nitrophenyl benzoate and p-nitrophenyl phenylacetate while that for the analogous hysdrocinnamate ester is unaffected. The results for the latter substrate are in accord with a general base-catalysed mechanism. Electron-withdrawal groups in the phenyl ring of phenyl acetates accelerate the enzyme acylation yielding a leaving group p of 2.05. The kinetic solvent isotope effects for acylation by p-nitrophenyl thiolacetate and by p-nitrophenyl acetate are close to 2.0. The mechanism of acylation of chymotrypsin by phenyl acetates is not unambiguously defined using these data.
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PMID:Mechanisms of acylation of chymotrypsin by phenyl esters of benzoic acid and acetic acid. 1 58

Activation of acetylated chymotrypsinogen with trypsin leads to catalytically active acetylated delta-chymotrypsin containing NH2-terminal isoleucine. The importance of the cationic terminus to the control of the active conformation of acetylated delta-chymotrypsin has been demonstrated (Oppenheimer, H. L., Labouesse, B., and Hess, G. P. (1966) J. Biol. Chem. 241, 2720). Later studies appeared to suggest that the modification of isoleucine-16 of delta-chymotrypsin is not accompanied by the loss of catalytic activity as measured by the hydrolysis of N-acetyl-L-tyrosine ethyl ester (Agarwal, S. P., Martin, C. J., Blair, T. T., and Marini, M.A. (1971)Biochem. Biophys. Res. Commun. 43, 510; Blair, T. T., Marini, M. A., Agarwal, S. P., and Martin, C. J. (1971) FEBS Lett. 1486) or by the loss of active site content (Ghelis, C., Garel, J. R., and Labouesse, J. (1970) Biochemistry 9, 3902). In the present studies, controlled acetylation of the terminal alpha-aminogroup of acetylated delta-chymotrypsin with acetic anhydride led to a progressive loss of active sites of the enzyme. Determination of the catalytic and kinetic properties of the modified enzyme with the specific ester substrate N-acetyl-L-tyrosine ethyl ester or the nonspecific substrates p-nitrophenyl acetate and cinnamyol imidazole gave nearly identical results. With N-acetyl-L-tyrosine ethyl ester as substrate, the Km (app) values for acetylated delta-chymotrypsin (1.0 plus or minus 0.1 mM) and the modified enzyme (0.67 plus or minus 0.05 mM) are nearly identical and the kcat value is reduced to about 25% in the latter enzyme species. This value correlates well with about 20% of the active sites in this enzyme as measured by the rapid initial liberation of p-nitrophenol. With p-nitrophenyl acetate as substrate, the acylation rate constants (0.13 plus or minus 0.04 s(-1) at pH 6.0, 25 degrees, in 3.3% acetonitrile) and the deacylation rate constants (0.01 s(-1) at pH 8.5, 25 degrees, in 3.3% acetonitrile) are identical for the acetyl isoleucine-16 and the isoleucine-16 enzymes. Furthermore, the residual enzyme activity could be correlated well with the residual NH2-terminal isoleucine content and with the moles of [1--14C]acetyl groups incorporated per mol of the enzyme. The activity associated with the modified enzyme can be attributed to the enzyme species in which isoleucine-16 of acetylated delta-chymotrypsin is not acetylated. These data are in general agreement with the studies of Ghelis et al. (1970) but are in disagreement with the results of Blair et al. (1971) and of Agarwal et al. (1971) and confirm the hypothesis that the final conformation of acetylated delta-chymotrypsin containing an acetylated NH2 terminus is catalytically inactive and resembles acetylated zymogen in many of its physical properties.
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PMID:Modification of isoleucine-16 acetylated delta-chymotrypsin. 114 Dec 36

Peptide synthesis was carried out in a variety of organic solvents with low contents of water. The enzyme was deposited on the support material, celite, from an aqueous buffer solution. After evaporation of the water the biocatalyst was suspended in the reaction mixtures. The chymotrypsin-catalyzed reaction between Z-Phe-OMe and Leu-NH2 was used as a model reaction. Under the conditions used ([Z-Phe-OMe]0 less than or equal to 40 mM, [Leu-NH2]0/([Z-Phe-OMe]0 = 1.5) the reaction was first order with respect to Z-Phe-OMe. Tris buffer, pH 7.8, was the best buffer to use in the preparation of the biocatalyst. In water-miscible solvents the reaction rate increased with increasing water content, but the final yield of peptide decreased due to the competing hydrolysis of Z-Phe-OMe. Among the water-miscible solvents, acetonitrile was the most suitable, giving 91% yield with 4% (by vol.) water. In water-immiscible solvents the reaction rate and the product distribution were little affected by water additions in the range between 0% and 2% (vol. %) in excess of water saturation. The reaction rates correlated well with the log P values of the solvent. The highest yield (93%) was obtained in ethyl acetate; in this solvent the reaction was also fast. Under most reaction conditions used the reaction product was stable; secondary hydrolysis of the peptide formed was normally negligible. The method presented is a combination of kinetically controlled peptide synthesis (giving high reaction rates) and thermodynamically controlled peptide synthesis (giving stable reaction products).
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PMID:Enzymatic peptide synthesis in organic media: a comparative study of water-miscible and water-immiscible solvent systems. 136 30

11 amino acid derivatives were tested as alpha-chymotrypsin substrates in the esterification reaction with methanol in organic media. The reactions were carried out in water-saturated ethyl acetate and in acetonitrile containing 4% water. alpha-Chymotrypsin adsorbed on Celite was used as a catalyst. From initial reaction rate measurements, the Michaelis-Menten parameters Vmax and KM were determined. All the amino acid derivatives tested were esterified, and the highest values of kcat/KM were obtained with the N-acylated aromatic amino acids. Correlations between Michaelis-Menten parameters and physical properties of the substrates such as molar refractivity (MR) and log P were deduced. The results show that the specificity of the alpha-chymotrypsin towards the side chain of the amino acids in organic media is the same as that in aqueous media. However, the specificity towards the N-protecting group is opposite to that in water, so the reaction medium affects the interaction of this part of the molecule with the enzyme to a large extent.
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PMID:Substrate specificity of alpha-chymotrypsin-catalyzed esterification in organic media. 176 79

Peptide synthesis with chymotrypsin in organic solvents was investigated and the apparent partition constants have been measured. We find that the Papp values of the most amino acids and peptide derivatives are drastically changed and the stereo- and regiospecificity in acetonitrile/water mixture is reduced.
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PMID:Chymotrypsin-catalyzed peptide synthesis in an acetonitrile-water-system: studies on the efficiency of nucleophiles. 182 34

The reactions were studied of N-acyl-L-amino acid esters with various D-amino acid amides catalyzed by free alpha-chymotrypsin, trypsin and proteinase K in acetonitrile containing 80 or 5 vol. % of water. In the medium with low water content the incorporation of D-amino acid amides into peptides proceeded with satisfactory yield sometimes approaching that of analogous L-L dipeptides. In the media with high water content negligible or low yields of L-D dipeptides were achieved. Synthesis of Boc-L-Trp-D-Phe-NH2 catalyzed by alpha-chymotrypsin was performed at molar ratio L: D = 3 : 2 in acetonitrile with 5 vol.% of water and the dipeptide was isolated in larger quantity. However, synthesis of the peptide bond did not occur at all when diastereomeric dipeptides having D-residue in the N-terminal P1' position were used even in the media with low water content.
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PMID:Serine proteinase-catalyzed incorporation of D-amino into model peptides in acetonitrile with low water content. 182 59

The 1H resonance assignments and secondary structure of the trypsin/chymotrypsin Bowman-Birk inhibitor from soybeans were determined by nuclear magnetic resonance spectroscopy (NMR) at 600 MHz in an 18% acetonitrile-d3/aqueous cosolvent. Resonances from 69 of 71 amino acids were assigned sequence specifically. Residues Q11-T15 form an antiparallel beta-sheet with residues Q21-S25 in the tryptic inhibitory domain and an analogous region of antiparallel sheet forms between residues S38-A42 and Q48-V52 in the chymotryptic inhibitory domain. The inhibitory sites of each fragment (K16-S17 for trypsin, L43-S44 for chymotrypsin) are each part of a type VI like turn at one end of their respective region of the antiparallel beta-sheet. These structural elements are compared to those found in other Bowman-Birk inhibitors.
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PMID:1H assignments and secondary structure determination of the soybean trypsin/chymotrypsin Bowman-Birk inhibitor. 201 1

A convenient approach to the synthesis of ring-substituted phenylalanine amides is described. Phase transfer alkylation of N-(diphenylmethylene)amino acetonitrile or N-(diphenylmethylene)glycine ethyl ester provides alpha-substituted imines 2 and 6. After acid hydrolysis and esterification, resolution with alpha-chymotrypsin provided 3'-substituted phenylalanine analogs 9a-d which could easily be converted to N alpha-t-butyloxycarbonyl-phenylalanine amides for use in the synthesis of peptide analogs. The approach described here is amenable to the synthesis of 3-substituted phenylalanines with a wide variety of substituents for determination of structure-activity relationships of peptides.
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PMID:Synthesis and resolution of novel 3'-substituted phenylalanine amides. 213 61

Exocrine proteins contained in human pancreatic juice were analyzed by reversed-phase high performance liquid chromatography (RP-HPLC). Pancreatic juice was saved by endoscopic retrograde cannulation of the main pancreatic duct in 17 persons: 12 without pancreatic disease, 3 patients suffering from recurrent acute pancreatitis probably due to pancreas divisum, 1 patient with a carcinoma of the pancreas, and 1 patient with chronic calcified pancreatitis. The juice proteins were separated on a silica column (Nucleosil 300-7 RP) by use of a multistep acetonitrile/water gradient (+0.1% trifluoroacetic acid). Up to 18 individual peaks could be separated by one analytical run (60 min). Molecular weight analysis by sodium dodecyl sulfate-gel electrophoresis indicated the presence of enzymes such as amylase, prophospholipase A2, procarboxypeptidases, trypsinogens, and chymotrypsinogens in certain peaks. Small residual enzymatic activities correlating with certain peaks were detected for amylase and chymotrypsin, and high residual activities were found for phospholipase A (recovery of enzymatic activity compared with the original sample amounted to 65%). Significant amounts of cathodic trypsin-like immunoreactivity were found in two certain peaks. By always loading 350 micrograms of protein/injection on the column the profiles of various samples showed similar patterns. Repeated injections of aliquots revealed highly reproducible profiles. RP-HPLC offers precise, reproducible, and rapid separation of the major proteins of human pancreatic juice.
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PMID:Resolution of human exocrine pancreatic juice proteins by reversed-phase high performance liquid chromatography (HPLC). 234 40

Protease-catalyzed peptide synthesis in acetonitrile/water mixtures, containing 0-90% water, was investigated. alpha-Chymotrypsin, as well as thermolysin, were deposited on solid supports, prior to exposure to the reaction media. Peptide syntheses were performed using both a kinetically controlled process (chymotrypsin) and an equilibrium-controlled synthesis (thermolysin). The activity of chymotrypsin decreased at low water contents. However, at low water contents (1-10%) hydrolytic side reactions were suppressed and high yields of dipeptides were obtained. Optimal water content for the thermolysin-catalyzed reaction was 4-8%. The dipeptides produced were fully soluble in the reaction mixtures. High operational stability for alpha-chymotrypsin was obtained during 216 h of reaction.
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PMID:The influence of water on protease-catalyzed peptide synthesis in acetonitrile/water mixtures. 305 21


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