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 tripeptide Z-GlyPheLeuNH2 was continuously synthesized in a high yield from three amino acid derivatives, Z-Gly, PheOMe, and LeuNH2, by immobilized thermolysin (IMT) and immobilized alpha-chymotrypsin (IMC) in an organic solvent, ethyl acetate. The optimal conditions for the synthesis of Z-GlyPheOMe were established theoretically. The yield of Z-GlyPheOMe with IMT in ethyl acetate saturated with buffer was more than 88% after continuous synthesis for 116 hr. The optimal conditions for the synthesis of Z-GlyPheLeuNH2 from Z-GlyPheOMe and LeuNH2 by IMC through transesterification was established in batch reaction experiments. When the concentration of water in the reaction solution was 17-20 microliters/ml, the activity of IMC was highest. The equilibrium between the water concentration in the reaction solution and that in the resin used for enzyme immobilization depended on the resin and was not affected by the presence of the enzyme immobilized. Z-GlyPheLeuNH2 was synthesized from Z-GlyPheOMe and LeuNH2 with a yield of 100%, by continuous reaction for 160 hr. The reactor for synthesis of this tripeptide was efficient and stable because of the use of transesterification and the choice of an appropriate organic solvent. The series plug-flow reactor was successfully operated for 220 hr with a yield of more than 80%. The residual activity of IMT was 94% and that of IMC was 100%.
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PMID:Continuous synthesis of a tripeptide by successive condensation and transesterification catalyzed by two immobilized proteinases in organic solvent. 136 63

The substrate specificities of alpha-chymotrypsin and subtilisins for peptide synthesis in hydrophilic organic solvents were investigated. Chymotrypsin exhibited high specificity to aromatic amino acids as acyl donors, while subtilisin Carlsberg and subtilisin BPN' were specific to aromatic and neutral aliphatic amino acids, in accordance with the S1 specificities of the enzymes for peptide hydrolysis in aqueous solutions. On the contrary, chymotrypsin exhibited higher specificities to hydrophilic amino acid amides as acyl acceptors (nucleophiles) for peptide synthesis with N-acetyl-L-tyrosine ethyl ester, in contrast to the S1' specificity for peptide hydrolysis and peptide synthesis in aqueous solutions. Furthermore, nucleophile specificity changed with the change in water-organic solvent composition; the increase in water content led to increase in relative reactivity of leucinamide to that of alaninamide. It was also found that protection of the carboxyl group of alanine by amidation is much preferable to protection by esterification in terms of reactivity as nucleophiles.
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PMID:Peptide synthesis by proteases in organic solvents: medium effect on substrate specificity. 136 70

Pressurization of enzyme-containing AOT-water-isooctane reversed micelles with low molecular weight gases leads to markedly different responses in activity characteristics. Microbial lipases exhibit a total cutoff in activity with as low a pressure as 2 MPa and a remarkable activity regain with depressurization. The observation also holds for reaction in monophasic organic solvents. The protease, alpha-chymotrypsin, is unaffected by pressurization until a critical pressure wherein micellar instability occurs. The use of pressure as a switch for lipase reaction in nonaqueous media is discussed.
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PMID:The use of pressure to modify enzyme activity in reversed micelles. 136 35

The total synthesis of the insect neuropeptide derivative Z-Gly-Gly-Ser-Leu-Tyr-Ser-Phe-Gly-Leu-NH2 has been carried out by a convergent solid phase strategy. For the coupling of the N-terminal pentapeptide to the C-terminal tetrapeptide, three different methods were assayed. Racemization of the acyl activated amino acid during the fragment condensation reaction was monitored by HPLC. Best results were obtained by enzymatic coupling in a low water containing media using adsorbed alpha-chymotrypsin. An optically pure product was obtained in 82% yield after 1 h of reaction. Chemical methods such as DIC/HOBt and BOP/HOBt/NMM always rendered highly optically impure products containing 10-20% of the D-epimer.
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PMID:Racemization free coupling of peptide segments. Synthesis of an insect neuropeptide. 139 72

By using solid-state NMR spectroscopy, the integrity of the active center of alpha-chymotrypsin was investigated under a variety of nonaqueous conditions. Specifically, 13C cross-polarization/magic angle spinning NMR was used to analyze the ability of alpha-chymotrypsin to stabilize a transition state intermediate analog after freezing, drying, and addition of organic solvents (both anhydrous and hydrated) to the resultant powder. Lyophilization disrupted 42 +/- 5% of the active centers; it was determined that this occurred during drying, as opposed to freezing. Seven anhydrous solvents caused 0-50% additional disruption, which occurred immediately on addition of the solvent to the enzyme powder. The extent of structural integrity loss correlated with the solvent hydrophobicity, indicating that further dehydration, i.e. stripping of water retained by the enzyme during lyophilization, was the cause. Enzyme samples prepared with lyoprotecting additives, sucrose and ammonium sulfate, exhibited varying degrees of stabilization against the drying step of lyophilization. Moreover, when hydrophilic anhydrous solvents, which had the highest propensity to strip bound water, were added to the resultant enzyme powders, no additional damage occurred.
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PMID:Solid-state NMR assessment of enzyme active center structure under nonaqueous conditions. 140 Mar 23

delta-Chymotrypsin has been alkylated by 1-13C- and 2-13C-enriched tosylphenylalanylchloromethane. In the intact inhibitor derivative, signals due to the 1-13C- and 2-13C-enriched carbon atoms have chemical shifts which titrate from 55.10 to 59.50 p.p.m. and from 99.10 to 103.66 p.p.m. respectively with similar pKa values of 8.99 and 8.85 respectively. These signals are assigned to a tetrahedral adduct formed between the hydroxy group of serine-195 and the inhibitor. An additional signal at 58.09 p.p.m. and at 204.85 p.p.m. in the 1-13C- and 2-13C-enzyme-inhibitor derivatives respectively does not titrate when the pH is changed and it is assigned to alkylated methionine-192. On denaturation/autolysis of the 1-13C-enriched enzyme-inhibitor derivative these signals associated with the intact inhibitor derivative are no longer detected, and a new signal, which titrates from 56.28 to 54.84 p.p.m. with a pKa of 5.26, is detected. The titration shift of this signal is assigned to the deprotonation of the imidazolium cation of alkylated histidine-57 in the denatured/autolysed enzyme-inhibitor derivative. Model compounds which form stable hydrates and hemiketals in aqueous solutions have been synthesized. By comparing the 13C titration shifts of these model compounds with those of the 13C enriched trypsin- and delta-chymotrypsin-inhibitor derivatives, we deduce that, in both of the intact enzyme-inhibitor derivatives, the zwitterionic tetrahedral adduct containing the imidazolium cation of histidine-57 and the hemiketal oxyanion predominates at alkaline pH values. It is estimated that in both the trypsin and delta-chymotrypsin-inhibitor derivatives the concentration of this zwitterionic tetrahedral adduct is 10,000-fold greater than it would be in water. We conclude that the pKa of the oxyanion of the hemiketal in the presence of the imidazolium cation of histidine-57 is 7.9 and 8.9 in the trypsin and delta-chymotrypsin-inhibitor derivatives respectively and that the pKa of the imidazolium cation of histidine-57 is greater than 7.9 and greater than 8.9 when the oxyanion is present as its conjugate acid, whereas, when the oxyanion is present, the pKa of the imidazolium cation is greater than 11 in both enzyme-inhibitor derivatives. We discuss how these enzymes preferentially stabilize zwitterionic tetrahedral adducts in the intact enzyme-inhibitor derivatives and how they could stabilize similar tetrahedral intermediates during catalysis. It is suggested that substrate binding could raise the pKa of the imidazolium cation of histidine-57 before tetrahedral-intermediate formation which would explain the enhanced nucleophilicity of the hydroxy group of serine-195.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:A study of the stabilization of tetrahedral adducts by trypsin and delta-chymotrypsin. 141 49

I.r. spectroscopy has been applied to the study of hydrogen-bonding of the unique ester carbonyl group of acylchymotrypsins in the oxyanion hole of the enzyme. This catalytic device provides electrophilic stabilization of negative charge in the transition states and tetrahedral intermediates along the reaction pathway. The use of 13C isotope substitution of the ester carbonyl group reinforces the previous observation [White & Wharton (1990) Biochem. J. 270, 627-637] that the ester carbonyl group is significantly polarized in the ground state by hydrogen bonding in the oxyanion hole. I.r. difference spectra of [carbonyl-12C]-minus [carbonyl-13C]-cinnamoyl-chymotrypsin as well as each of these acylenzymes minus free enzyme are reported. These spectra show that the contribution of protein perturbation (i.e. spectral features that arise from the enzyme which is distorted on acylation) in [carbonyl-12C]cinnamoyl-chymotrypsin minus free enzyme spectra is significant. The contribution of the perturbation components of the spectra is pH-dependent and can represent up to 50% of the total absorbance in the spectral region from 1690 to 1740 cm-1. Use of the isotopic difference method has allowed problems associated with protein perturbation to be eliminated. Similar difference spectra are presented for dihydrocinnamoyl-chymotrypsin. In this case the effect of perturbation is very marked and leads to the cancellation of the band assigned to the non-bonded conformation of the acyl group which has previously only been observed at higher pH. The isotopic difference method again proves reliable and shows that the frequency difference previously used to calculate the ground-state electronic strain induced by the oxyanion-hole catalytic device is not affected by the perturbation, although the amplitudes of the spectral features are different. A study of the deacylation of cinnamoyl-chymotrypsin in water and deuterium oxide using both u.v. and i.r. spectroscopies has confirmed that the use of deuterium oxide as solvent has no serious effect on the deacylation behaviour of the enzyme. I.r. bands assigned to nonproductive and productive conformers decline identically during deacylation, which shows that the conformers are in dynamic exchange on the reaction time-scale.
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PMID:Analysis and elimination of protein perturbation in infrared difference spectra of acyl-chymotrypsin ester carbonyl groups by using 13C isotopic substitution. 141 85

We have investigated the ability of Bowman-Birk inhibitor, a protease (trypsin and chymotrypsin) inhibitor, to protect against radiation-induced thymic lymphosarcoma in C57Bl/6NCr1BR mice. Fifty-five 7-week-old male mice were randomized into 11 groups and gavaged 5 days per week with purified Bowman-Birk inhibitor, Bowman-Birk inhibitor concentrate, and autoclaved Bowman-Birk inhibitor concentrate. Following 7 days of gavage, those mice undergoing total-body or sham total-body irradiation received 1.7 Gy weekly for 4 weeks. At 6 months following the radiation exposure, all mice were sacrificed and examined histopathologically. Samples of Bowman-Birk inhibitor concentrate, purified Bowman-Birk inhibitor, and autoclaved Bowman-Birk inhibitor concentrate were evaluated with thin-layer chromatography. The mice treated with total-body irradiation and autoclaved Bowman-Birk inhibitor had significantly (P < 0.05) fewer deaths, lower average grade of lymphosarcoma, and larger fat stores compared to those treated with total-body irradiation and water gavage. The results for the total-body-irradiated mice receiving Bowman-Birk inhibitor concentrate suggested an effect midway between these two groups. Thin-layer chromatography analysis indicated that sterols and the phospholipids varied in the three different samples in a way that approximately corresponded with the observed effects. We have observed that an autoclave-resistant factor in soybeans is capable of reducing metastasis of radiation-induced lymphosarcoma and weight loss in C57Bl/6NCr1BR mice, presumably by preventing the extension and metastasis of cancer cells. Thus, in addition to the anticarcinogenic Bowman-Birk inhibitor, there appears to be another anticarcinogenic agent in soybeans which is capable of inhibiting the later stages of cancer cell development.
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PMID:Protection against metastasis of radiation-induced thymic lymphosarcoma and weight loss in C57Bl/6NCr1BR mice by an autoclave-resistant factor present in soybeans. 143 9

The relationship between physicochemical properties and oral absorption was investigated in prodrugs for the oral delivery of disodium cromoglycate (DSCG). To improve the lipophilicity of DSCG, various lipophilic moieties were introduced into the twin carboxyl groups. However, this did not lead to improved oral absorption in rabbits because of loss of water solubility, in spite of improved lipophilicity. Water-soluble prodrugs, in which an amino acid was introduced into a hydroxy group by ester linkage in addition to ethyl residues at twin carboxyl groups of DSCG, were synthesized and examined for oral absorption in rabbits and rats. The oral absorption of these prodrugs was affected by the species of amino acids introduced as a water-soluble moiety. Therefore, we examined the relation between the oral absorption of water-soluble prodrugs and the hydrolysis rate of the amino acid moiety. A good linear correlation was obtained between the oral absorption and the hydrolysis rate constant catalyzed by digestive enzymes, trypsin or alpha-chymotrypsin. It was thus concluded that the amino acid moiety of water-soluble prodrugs must be rapidly hydrolyzed to a permeable lipophilic prodrug still possessing the ethyl moiety at twin carboxyl groups in the small intestinal tract for good oral absorption.
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PMID:Importance of hydrolysis of amino acid moiety in water-soluble prodrugs of disodium cromoglycate for increased oral bioavailability. 144 81

At the aim of investigating whether the early rapid phase of enzyme turnover is different in reverse micelles compared with bulk water, the kinetic properties of alpha-chymotrypsin have been studied in reverse micelles formed by sodium bis(2-ethylhexyl)sulfosuccinate in isooctane. Pre-steady state and steady-state kinetic constants, in water and in reverse micelles, have been determined by stopped-flow spectrophotometry for the hydrolysis of two substrates, namely acetyl-L-tryptophan-p-nitrophenyl ester and p-nitrophenyl acetate. It has been shown that, for both substrates, the acylation rate constant (k2) is very much lower in reverse micelles than in water. However, the deacylation rate constant (k3) and the turnover number (kcat) are not significantly changed in reverse micelles with respect to bulk water. Therefore, despite considerable rate changes in the acylation step, deacylation is rate limiting both in water as well as in reverse micelles, under the experimental conditions used.
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PMID:Kinetic behaviour of alpha-chymotrypsin in reverse micelles. A stopped-flow study. 151 84


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