Inhibition Of Α-chymotrypsin Research Articles

Reformable intramolecular cross‐linking of the N‐terminal domain of heparin cofactor II

The crystal structure of a heparin cofactor II (HCII)-thrombin Michaelis complex has revealed extensive contacts encompassing the N-terminal domain of HCII and exosite I of the proteinase. In contrast, the location of the N-terminal extension in the uncomplexed inhibitor was unclear. Using a disulfide cross-linking strategy, we demonstrate that at least three different sites (positions 52, 54 and 68) within the N terminus may be tethered in a reformable manner to position 195 in the loop region between helix D and strand s2A of the HCII molecule, suggesting that the N-terminal domain may interact with the inhibitor scaffold in a permissive manner. Cross-linking of the N terminus to the HCII body does not strongly affect the inhibition of alpha-chymotrypsin, indicating that the reactive site loop sequences of the engineered inhibitor variants, required for interaction with one of the HCII target enzymes, are normally accessible. In contrast, intramolecular tethering of the N-terminal extension results in a drastic decrease of alpha-thrombin inhibitory activity, both in the presence and in the absence of glycosaminoglycans. Treatment with dithiothreitol and iodoacetamide restores activity towards alpha-thrombin, suggesting that release of the N terminus of HCII is an important component of the multistep interaction between the inhibitor and alpha-thrombin.

Inhibition of α-chymotrypsin with thiol-bearing substrate analogues in the presence of zinc ion

We have demonstrated that thiol-bearing analogues of α-chymotrysin (α-CT) substrates such as ( S)-(1-benzyl-2-thiolethyl)-carbamic acid, benzyl ester ( 3) inhibits α-CT, a prototypical serine protease, in the presence of Zn(II) ion. They constitute a novel class of small molecule inhibitors for α-CT believed to inhibit the enzyme by forming a ternary complex consisting of α-CT, Zn(II) ion, and the inhibitor.

Structural approach of the mechanism of inhibition of α-chymotrypsin by coumarins

A pharmacophore associated to the inhibiton of α-chymotrypsin has been built based on the structural and electronic characterization of a series of coumarin derivatives.

Development and Analysis of a Polymer Protecting from Luminal Enzymatic Degradation Caused by α-Chymotrypsin

We have been developing a modified polymer which protects inserted peptide and protein drugs from chymotrypsinic degradation. A trypsin-chymotiypsin-inhibitor was covalently bound to a bioadhesive polymer (poly (acrylic acid)) by a condensation reaction of the available amino groups of the inhibitor with the carboxyl groups of the polymer. The inhibition of α-chymotrypsin by the isolated conjugate was determined by an enzyme assay using N-benzoyl-L-tyrosine ethyl ester as substrate. It had an inhibitory effect of 2.4 CIU (= chymotrypsin inhibiting units) per mg. The protective effect was also evaluated in a drug delivery system containing 5% of this modified polymer. Under physiological α-chymotrypsin concentrations it demonstrated, in lateral parts of the carrier matrix, the remaining of 71.4% ± 14.3% (means of three experiments; ± S.D.) of undigested inserted test-protein (β-galactosidase) afer 7 hr of incubation at 37 ± 0.5°C. Samples from the center of the matrix did not show any digestion of the protein. On the other hand a complete digestion in lateral parts and the remaining of only 67% ± 14.4% of undigested test-protein in the center, could be observed by the same delivery system without the conjugate. Because of this verified protective effect and the possibility to exclude toxic side effects of the inhibitor by immobilization, the system described here seems to be an important step towards a successful peroral (poly)peptide drug administration.

The Inhibition of α-chymotrypsin predicted using theoretically derived molecular properties

The structures and molecular properties of 95 aromatic and heteroaromatic ligands previously tested as reversible inhibitors of chymotrypsin catalysis have been calculated using AM1. The properties obtained have been used as input for multiple linear regression analysis and as descriptors for a back-propagation neural network to predict the binding affinity of α-chymotrypsin inhibitors. Using polarizability, molecular shape, electrostatic similarity, dipole moment, ClogP, and the diagonalized quadrupole moments of the ligands, correlation coefficients between calculated and experimental affinities of 0.96 for the training set and 0.89 for the test set were obtained using a neural network. The performance of the multiple linear regression was significantly worse, although useful QSARs were also obtained.

Probing enzyme stereospecificity. Inhibition of α-chymotrypsin and subtilisin Carlsberg by chiral amine- and aminoalcohol-derivatives

Various enantiomeric amine and aminoalcohol amide and α-ketoamide derivatives have been evaluated as competitive inhibitors of the representative serine proteases α-chymotrypsin (CT) and subtilisin Carlsberg (SC). Each compound studied was an effective competitive inhibitor of both enzymes. However, only for the best inhibitor, N-pyruvoyl-1-(1-naphthyl)ethylamine (K1 27 μM for the S-enantiomer with CT), was noteworthy enantiomeric discrimination manifest, with the S-enantiomer being a significantly more powerful inhibitor of CT and SC than its R-counterpart by factors of 12.6- and 73-fold, respectively. The enzyme-inhibitor interactions responsible for this strong binding and enantiomeric discrimination were revealed by molecular modelling analyses.

Inhibition of α-Chymotrypsin with an Enzyme-Activated N-Nitrosoamide: Active-Site Labeling by the Naphthylmethyl Cation

α-Chymotrypsin was irreversibly inhibited with an enzyme-activated N-nitrosoamide inhibitor, N-nitroso-N-(1-naphthylmethyl)-N′-isobutyrylalanine; alkylation of the active-site residues by the naphthylmethyl cation produced in the enzymatic reaction occurred. The inhibited enzyme was reduced and aminoethylated and then subjected to tryptic and chymotryptic digestion. Separation of the digest by reversed-phase HPLC revealed one major new peak relative to that of a control run from the native enzyme. Subsequent amino acid analysis and sequencing along with fast atom bombardment-mass spectrometry measurements indicated that this new peak stemmed from an active-site peptide, Met-192-Leu-199, and that the naphthylmethyl label was attached to the side-chain oxygen of Ser-195. The general approach employed can be applied to labeling active sites of a variety of hydrolytic enzymes.

Cerebrospinal Fluid of Patients with Senile Dementia of Alzheimerʼs Type Shows an Increased Inhibition of α-Chymotrypsin

The inhibition of alpha-chymotrypsin induced by cerebrospinal fluid from patients with senile dementia of Alzheimer's type, vascular dementia, and nondemented controls was investigated. We optimized the sensitivity of the assay so that the inhibition of alpha-chymotrypsin could be measured in all samples. The competitive inhibition was proportional to the amount of cerebrospinal fluid (CSF) added to the reaction mixture. After correction for protein concentration, the inhibition was higher with CSF from patients with senile dementia of Alzheimer's type than with those from patients with vascular dementia or controls. Inhibitory activity appeared to be specific for alpha-chymotrypsin because no inhibition for papain was found. Moreover, the depletion of alpha 1-antichymotrypsin from CSF by immunoadsorption revealed that this serpin was responsible for the disappearance of the inhibitory activity. Our findings suggested that the increased alpha-chymotrypsin inhibitory activity might represent an in vivo functional index of an abnormal protease metabolism in patients with Alzheimer's disease.

Purification and characterization of a fungal protease specific protein inhibitor (FPI-F) in the silkworm haemolymph

1. 1. One of fungal protease inhibitors (FPI-F) in the haemolymph of the silkworm, Bombyx mori, was purified by ammonium sulphate precipitation, acid and heat treatments, cation exchange chromatography on a CM-Toyopearl and hydrophobic chromatography on a butyl-Toyopearl column. About 2000-fold purification was achieved and 13% of the activity was recovered. 2. 2. The inhibitor had a mol. wt of approx. 6000. The kinetic analysis showed that FPI-F inhibits fungal protease uncompetitively ( K i = 8.6 × 10 -8 M) and formed equimolar complex with fungal protease. 3. 3. FPI-F suppressed strongly fungal proteases and subtilisin but the inhibition of α-chymotrypsin and trypsin was extremely weak.

Physical aspects of the inhibition of enzymes by hydrocarbons: The inhibition of α-chymotrypsin by chlorinated aromatics and alkanes

The inhibition of α-chymotrypsin by a series of chlorinated hydrocarbons, including polychlorinated biphenyls, has been studied. The solubility of the hydrocarbons was determined by autocorrelation analysis of light scattering. Kinetic analysis indicated that inhibition of the enzyme occurs when 1–2 molecules of inhibitor bind per molecule of enzyme. Chlorinated aromatics including polychlorinated biphenyls were more potent than monochloroalkanes in inhibition of the enzyme. Considerable inhibition was seen when some compounds were present as micelles. Molar volume correlations suggest that chlorinated hydrocarbons exert effects on soluble enzymes similar to their effects on membrane-bound enzymes, and that a membrane lipid phase is not essential for this type of inhibition.

Inhibition of α-chymotrypsin by diethyl [formula omitted] phosphate: An undergraduate experiment

Inhibition of α-chymotrypsin by diethyl [formula omitted] phosphate: An undergraduate experiment

Inhibition of α-chymotrypsin by hydroxymethyl analogues of d- and [formula omitted] and N-acetyltryptophan of potential affinity labeling value

d- and l -N- acetyl - and N, O-diacetylphenylalaninol and -tryptophanol are found to be competitive inhibitors of α-chymotrypsin with K 1 values in the range 4.3–65.7 mM. In each case, the N,O-diacetates are better inhibitors than the corresponding hydroxymethyl compound and although hydrogen bonding of hydroxymethyl groups at the nucleophilic site ( n) does contribute to binding, interactions of O-acetate functions with this region are much more significant. The tryptophanol derivatives are more effective by a factor of about 5 than those of phenylalaninol and d enantiomers bind approximately twice as strongly as their l counterparts. The results obtained indicate that phenylalaninol, or preferably tryptophanol, structures of the types studied are suitable for use as alkylating group carriers in affinity labelling experiments designed to probe the more remote amino acid residues of the n-region of the active site. They are also of potential value for X-ray and NMR studies on the differences between the binding of d and l enantiomers by α-chymotrypsin.

Specificity-determining site of alpha-chymotrypsin. 3. Inactivation of alpha-chymotrypsin by p-diazo-N-acetyl-L-phenylalanine methyl ester.

The diazo compound derived from Nα-acetyl-L-phenylalanine methyl ester, DAPM (p-diazo-N-acetyl-L-phenylalanine methyl ester), has been synthesized and tested as an active site-directed irreversible inhibitor of α-chymotrypsin [EC 3.4.4.5]. DAPM at 50-fold molar excess causes 60% inhibition of α-chymotrypsin (2×10−5M) in about 5 hr at pH 8.0 and 4°C. β-Phenylpropionic acid completely protects α-chymotrypsin from inactivation by this reagent, indicating that azo-coupling occurs probably at or near the active center. From the results of an efficiency assay using specific substrates and all-or-none assay using cinnamoylimidazole, the remaining activity of partially inactivated enzyme was shown to depend only on the active enzyme molecules present in the preparation, whose active sites could not be modified with the diazo reagent. Amino acid analysis and spectral analysis of the modified chymotrypsin indicate that azotization by DAPM occurs only in tyrosine and lysine residues, and not in histidine, methionine, and serine residues which have been reported to be present in the active site of this enzyme. The reactivity toward azotization of tyrosine-146, the carboxyl terminal residue of the B chain of α-chymotrypsin, by DAPM was also examined. The results showed that either in the presence of a reversible competitive inhibitor or in its absence, tyrosine-146 is more readily accessible to attack by the reagent as compared with not only the three other tyrosine residues but also any other amino acid residues in α-chymotrypsin. However, further study is required to identify the amino acid residue modified with DAPM, which might be the site of inactivation of α-chymotrypsin by attack of the reagent and be involved in the formation of the specificity-detoermining site of the enzyme.

Naturally occurring crystals in the potato: Isolation and identification as a protein

Cubical crystals occurring naturally in potato tubers were isolated and identified as protein. The amino acid composition was characterized by a relatively large amount of lysine and a near absence of cysteine/cystine. The major electrophoretic component had an approximate molecular weight of 77,000. The preparation exhibited trace proteolytic activity and a weak inhibition of α-chymotrypsin.

Inhibition of α-Chymotrypsin by Diethyl Ether and Certain Alcohols: a New Type of Competitive Inhibition

The enzyme used was a salt-free, twice crystallized sample of cu-chymotrypsin supplied by the Sigma Chemical Company. The methyl ester of hydrocinnamic acid (3-phenylpropionic acid) was prepared by refluxing 5 g of the acid with 750 ml of Fisher’s chemically pure methanol and a few drops of concentrated sulfuric acid as a catalyst. The reaction mixture was refluxed for 2 days. The excess of methanol was evaporated under reduced pressure, and the residue was extracted with ether. The ether was washed with 10 100 ml portions of 5% sodium bicarbonate. The ether was evaporated under reduced pressure, and the ester was distilled under reduced pressure, the middle 30% being collected. The boiling poin$ was 236-237” at atmospheric pressure. The methyl hippurate was prepared by heating a mixture of hippuric acid in the presence of a large excess of methanol to 190” under an atmosphere of hydrogen at approximately 2500 pounds per square inch (3). After subsequent purification, the melting point was 82-83”. The concentration of the enzyme was approximately 1