The stereospecificity of α-chymotrypsin

1. The rates of deacylation of acyl-alpha-chymotrypsins in which the hydrogen-bonding capacity of the acylamino group of the substrate has been systematically removed were measured. 2. The ratio of deacylation rates of l- and d-acyl-enzymes is found to depend largely on the existence in the substrate of an amido -NH- group. 3. The data presented agree with the postulate that the stereospecificity of alpha-chymotrypsin is exercised in catalytic rather than binding steps, and that the active site of the enzyme presents three loci to the substrate: the site containing the catalytic functionalities (including serine-195), the hydrophobic area for amino acid side-chain binding, and a hydrogen-bond acceptor site for acylamino group binding. 4. It is noted that, though the hydrogen-bonding site is crucial for the stereospecificity, the free energy of binding of substrates and inhibitors is dominated by the hydrophobic interaction. 5. It is tentatively proposed that alpha-chymotrypsin selects a high-energy conformation of the substrate when the latter binds at the enzyme's active site.     

Specificity and stereospecificity of α-chymotrypsin

1. The optically pure p-nitrophenyl esters of the d and l enantiomers of N-acetyl-tryptophan, N-acetylphenylalanine and N-acetyl-leucine, and the p-nitrophenyl ester of N-acetylglycine, have been prepared. 2. These materials are all substrates of α-chymotrypsin, and the rates of deacylation of the corresponding acyl-α-chymotrypsins have been determined. 3. As the size of the amino acid side chain increases, the l series deacylate progressively faster than the N-acetylglycyl-enzyme, and the d series progressively more slowly. 4. The results are interpreted in terms of a three-locus model of the enzyme's active site, which accounts for the interrelationship between substrate specificity and stereospecificity observed. 5. The concepts of negative specificity and of specificity saturation are introduced.     

The binding of inhibitors to α-chymotrypsin

1. The binding of three competitive inhibitors, N-acetyl-d-tryptophan, N-acetyl-l-tryptophan and N-acetyl-d-tryptophan amide, to alpha-chymotrypsin was studied over the pH range 2.20-9.65 by the technique of equilibrium dialysis. 2. Within the limits of the experimental method, the binding of the uncharged amide inhibitor is independent of pH over the range investigated. 3. The binding of each of the enantiomeric acids is dependent on the ionization of a group on the free enzyme, of apparent pK(a)7.3. 4. It is shown that the ionizing group results in the active site of the enzyme developing a net negative charge above pH7.3. 5. The enzyme groups responsible are tentatively identified, and the significance of the binding constants with respect to the enzymic catalysis is discussed.     

Thermostability of α-chymotrypsin encapsulated in reversed micelles

Summary The influence of water content, additives, pH and substrate concentration on the thermostability of -chymotrypsin entrapped in a reversed micellar system of the cationic surfactant TTAB/heptane/ chloroform, was studied. Increasing the water level inside the reversed micelles diminishes the enzyme stability. Enzyme stability enhancement was achieved with the addition of glycerol, by increasing the nucleophile concentration or by decreasing the buffer pH.     

Amino acid ester synthesis by immobilized α-chymotrypsin

Summary A screening of immobilized -chymotrypsin preparations suitable for the synthesis of N-acetyl-L-tyrosine ethyl ester from N-acetyl-L-tyrosine and up to 99% ethanol was carried out. -Chymotrypsin adsorbed to Sepharose LH-20 or covalently bound to Sepharose 4B (tresyl chloride activation) was found to be an efficient catalyst. A column packed with immobilized enzyme retained 60% of its initial activity after 6 days of operation in a cyclohexane-ethanol medium.     

Stability of immobilized α-chymotrypsin in supercritical carbon dioxide

Summary Inactivation of immobilized -chymotrypsin in supercritical carbon dioxide was with a first-order kinetic behaviour. The increase in either the pressure or the temperature of the fluid enhanced the inactivation process of the enzyme. The fluid density was shown as a key parameter on the enzyme stability, enhancing the half-life time proportionally to the physical phase of CO₂, as follows: liquid > supercritical > gas. However, the number of pressurization/depressurization cycles, and the water content of the derivative increased greatly the loss of activity.     

L-Dopa ester synthesis catalyzed by α-chymotrypsin in organic solvents

Summary Esters of N-unprotected amino acids have been shown to be suitable substrates for -chymotrypsin-catalyzed reactions in organic solvents. Based on this observation, a wide range of L-Dopa esters were synthesized via enzymic transesterification, and up to 50% yields were obtained using various alcohols as acyl acceptors.     

Separation of trypsin from trypsin α-chymotrypsin mixture by affinity-ultrafiltration

Summary Water-sobuble trypsin specific macroligands were prepared to separate the trypsin -chymotrypsin mixture with affinity-ultrafiltration technique. Soya bean trypsin inhibitor (STI) attached to cyanogen bromideactivated Dextran showed a good selectivity and low non-specific adsorption properties. The experiments performed with STI-Dextran polymer gave a 81% purified trypsin from 50%-50% mixture.     

Thermostability of α-chymotrypsin in water/organic solvent systems

Summary The influence of pH, temperature, substrate concentration and organic solvents (dimethylformamide, dimethylsulfoxide) on the -chymotrypsin stability in a water/organic solvent system was studied. The enzyme activity was measured as the dipeptide, AcPheLeuNH₂ synthesis and the ester substrate hydrolysis. Enzyme stability was enhanced by lower pH and temperature values and higher substrate concentrations. Dimethylsulfoxide allowed an higher enzyme stability than dimethylformamide. -Chymotrypsin displayed an higher stability in the water medium when it was compared to the organic system.     

Effect of aluminum on the binding properties of α-chymotrypsin

 The effect of aluminum ions on the binding properties of α-chymotrypsin has been studied. The results show that aluminum does not affect the catalytic rate constant k _cat, but it acts as an enzyme activator favoring the binding of the substrate to the catalytic site (i.e. decreasing K _m). Furthermore, aluminum binding to α-chymotrypsin displays about a threefold decrease in its affinity for the macromolecular inhibitor bovine pancreatic trypsin inhibitor (BPTI). Altogether, the different effect of aluminum on the binding of synthetic substrates (e.g. N-α-benzoyl-l-tyrosine ethyl ester, BTEE) and macromolecular inhibitors (e.g. BPTI) to α-chymotrypsin suggests the occurrence of an aluminum-linked conformational change in the enzyme molecule which brings about a marked structural change at the primary and secondary recognition sites for substrates and inhibitors. The modulative effect exerted by aluminum on the enzyme hydrolytic activity has been investigated also as a function of pH. The ion-linked effect appears to be dependent on the pH in a complex fashion, which suggests that aluminum binding is controlled by the protonation of at least two classes of residues on the enzyme molecule. Received: 5 December 1996 / Accepted: 11 March 1997     

Angiotensin I-converting enzyme inhibitory oligo-tyrosine peptides synthesized by α-chymotrypsin

Oligo-tyrosine peptides with degrees of polymerization ranging from 2 to 5 could be synthesized by α-chymotrypsin-catalyzed reaction with l-tyrosine ethyl ester in aqueous media, although the peptide yield was low due to a preferential hydrolysis of the substrate. It was also confirmed that α-chymotrypsin efficiently converted tyrosine tetramer to the dimer which was resistant to the digestion. Both Tyr-Tyr and Tyr-Tyr-Tyr showed high inhibitory activity for angiotensin I-converting enzyme from rabbit lung, and their IC₅₀ values were 34 μM and 51 μM, respectively. These two peptides exhibited a mix of competitive and noncompetitive inhibitions. Tyr-Tyr-Tyr was first recognized as an ACE inhibitor, suggesting that α-chymotrypsin could be applied to synthesis of novel potential materials for antihypertensive medicines.     

The binding of inhibitors to α-chymotrypsin at alkaline pH

1. The binding of the competitive inhibitor N-acetyl-d-tryptophan amide to alpha-chymotrypsin has now been studied at pH values up to 10.6, by the technique of equilibrium dialysis. 2. This binding depends on the ionization of a group on the free enzyme with apparent pK(a) 9.3 at 5 degrees . 3. This group is tentatively identified as that responsible for an enzyme conformation change at high pH values, on which the catalytic activity of the enzyme also depends.     

Synthesis of kyotorphin precursor from eutectic mixtures catalysed by α-chymotrypsin

The kyotorphin precursor, N-carbobenzoxyl-l-tyrosine-l-arginine amide (N-CBZ-l-Tyr-l-ArgNH₂), was synthesized from N-CBZ-l-tyrosine ethyl ester (N-CBZ-l-TyrOEt) and l-arginine amide (l-ArgNH₂) by using -chymotrypsin. Eutectic mixtures were formed by mixing the substrates in the presence of water and/or organic solvents as adjuvants. The eutectic temperature was obtained with a 0.45 l-ArgNH₂ mole fraction. It was lowered to 23 °C by addition of 10% (v/w) water and 5% (v/w) dimethyl formamide, thus maintaining homogeneous liquid states at the reaction temperature of 30 °C. After 9 h the solutions became solidified and no further reaction took place. Approximately 90% (mol/mol) conversion was achieved from the substrate mixtures with substrate contents responsible for more than 80% (w/w) of the total mixture.     

Anticancer and α-chymotrypsin inhibiting diterpenes and triterpenes from Salvia leriifolia

Salvialeriol (1), a new abietane-type diterpene, was isolated from Salvia leriifolia Benth. (Salvia leriaefolia), along with two known abietane-type diterpenoids, 6-hydroxysalvinolone (2) and deacetylnemorone (3), and two known triterpenes, 2-acetoxylupeol (4), and lupine-2,3-diol (5). Compounds 2–5 are reported here for the first time from this species. Compound 4 was previously reported as a synthetic derivative of 5 and this is the first report of its isolation from a natural source. Compounds 2, 3 and 5 exhibited a potent antiproliferative activity against the prostate cancer cell lines (PC3) with IC₅₀ of 3.9 ± 0.1, 6.2 ± 0.1 and 2.8 ± 0.1 μM, respectively, and cervical cancer cell lines (HeLa) with IC₅₀ of 8.0 ± 0.3, 2.6 ± 0.1 and 2.7 ± 0.1 μM, respectively. Whereas compounds 1 and 4 showed moderate antiproliferative activities against the cell lines. Compounds 1–5 were also evaluated for the inhibition of α-chymotrypsin, a protease enzyme, and 2 exhibited a competitive inhibition of the enzyme (IC₅₀ = 188.8 μM).     

An examination of the utility of photogenerated reagents by using α-chymotrypsin

As a test of the labelling characteristics of photogenerated reagents, an aryl azide was photolysed in the aromatic-binding locus of a protein of known tertiary structure. The acyl-enzyme derived from the reaction of alpha-chymotrypsin with the p-nitrophenyl ester of p-azido[(14)C]cinnamate was isolated and photolysed. About 60% of the acyl group is covalently bound to the protein after photolysis and deacylation, and labelled enzyme is inactive. The covalently attached label is localized in the C chain of chymotrypsin, and there are firm indications that the major labelled tryptic fragment of the C chain is that which constitutes the aromatic-binding locus of the enzyme. The high degree of labelling of that portion of the protein molecule predicted on the basis of the known chemistry and structure of alpha-chymotrypsin, provides gratifying confirmation of the utility of the photo-labelling method.     

Time dependent behaviour of the cross-linking reaction of α-chymotrypsin with glutaraldehyde

Summary Immobilization of -chymotrypsin was done by cross-linking the enzyme with glutaraldehyde. Studies were done in a batch reactor to find out the effects of the concentrations of -chymotrypsin and glutaraldehyde on the rate of particle formation, the final yield of the crosslinked particles, and the residual activity of the cross-linked enzyme. Both intra- and inter-molecular cross-linking play important role at different time course of the reaction. At the begining of the cross-linking reaction, the large drop in the activity is because of the intra-molecular cross-linking and later on the large increase in the particle formation is because of the inter-molecular cross-linking.     

Thermal stability of immobilized α-chymotrypsin is additionally increased by chaotropic compounds

Summary Temperature dependence of the rate constant of irreversible thermal inactivation, k_in, of immobilized -chymotrypsin depends markedly on the number of covalent bonds between the enzyme and support. When the number of bonds is big enough (thirteen), the dependence is linear as presented in Arrhenius plot (log k_in versus reciprocal temperature). However, if the number of such bonds is moderate or small (six or two), the temperature dependence of k_in, has a pronounced zig-zag character. This difference in the inactivation behaviour is attributed to an ability of moderately or mildly attached -chymotrypsins to accomplish a transition into a less ordered, catalytically inactive conformation and to inability of rigidly bound enzyme to pass such a transition. Chaotropic salts additionally stabilize this loose conformation of mildly or moderately bound -chymotrypsins against irreversible thermal inactivation but are without effect on the stability of rigidly bound enzyme.     

Synthesis and in vitro α-chymotrypsin inhibitory activity of 6-chlorobenzimidazole derivatives

A library of benzimidazole derivatives 1–20 were synthesized, and studied for their α-chymotrypsin (α-CT) inhibitory activity in vitro. Kinetics and molecular docking studies were performed to identify the type of inhibition. Compound 1 was found to be a good inhibitor of α-chymotrypsin enzyme (IC₅₀ = 14.8 ± 0.1 μM, K_i = 16.4 μM), when compared with standard chymostatin (IC₅₀ = 5.7 ± 0.13 μM). Compounds 2–8, 15, 17, and 18 showed significant inhibitory activities. All the inhibitors were found to be competitive inhibitors, except compound 17, which was a mixed type inhibitor. The substituents (R) in para and ortho positions of phenyl ring B, apparently played a key role in the inhibitory potential of the series. Compounds 1–20 were also studied for their cytotoxicity profile by using 3T3 mouse fibroblast cells and compounds 3, 5, 6, 8, 12–14, 16, 17, 19, and 20 were found to be cytotoxic. Molecular docking was performed on the most active members of the series in comparison to the standard compound, chymostatin, to identify the most likely binding modes. The compounds reported here can serve as templates for further studies for new inhibitors of α-chymotrypsin and other chymotrypsin-like serine proteases enzymes.

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The interaction of α-chymotrypsin with isosteric substrates of different charge type

1. The synthesis of three substrates of alpha-chymotrypsin of closely similar steric requirements but different charge type is reported. 2. The interaction of these compounds [SS-dimethyl-(l-3-carboxymethyl-3-acetamido)propyl sulphonium iodide, l-2-acetamido-5-methylhexanoic acid methyl ester and N-acetyl-l-glutamic acid alpha-methyl ester] with alpha-chymotrypsin has been studied. 3. For the charged substrates, values of k(0) are two orders of magnitude smaller than, and values of K(m) two orders of magnitude larger than, the corresponding values for the uncharged isostere. 4. The results are interpreted in terms of the known specificity of the enzyme, and the relationship between binding and kinetic specificities is discussed.