Catalytic activity and denaturation of enzymes in water/organic cosolvent mixtures. Alpha-chymotrypsin and laccase in mixed water/alcohol, water/glycol and water/formamide solvents

The dependence of the catalytic activities of alpha-chymotrypsin and laccase on the concentration of organic cosolvents (alcohols, glycols and formamides) in mixed aqueous media has a pronounced threshold character: it does not change up to a critical concentration of the non-aqueous cosolvents added, yet further increase of the latter (by only a small percentage, by vol.) leads to an abrupt decrease in enzyme activity. Fluorescence studies indicate that the inactivation results from reversible conformational changes (denaturation) of the enzymes. There is a linear correlation between the critical concentration of residual water (at which the enzyme inactivation occurs in a threshold manner) and the hydrophobicity of the organic cosolvents added. A quantitative criterion is suggested for the selection of organic cosolvents to be used for enzymatic reactions in homogeneous water/organic solvent media.     

Synthesis of N-protected peptide alcohols catalyzed by subtilisin or alpha-chymotrypsin in organic solvents

A series of N-protected peptide alcohols were synthesized using amino alcohols with unprotected hydroxy groups as amino components by the catalysis of subtilisin or alpha-chymotrypsin in organic solvents. N-protected aromatic amino acid esters were more suitable as acyl donors for subtilisin. The influences of different N-protecting groups, organic solvents, and content of water on synthesis of N-protected peptide alcohols were systematically studied.     

Sol-gel immobilization of serine proteases for application in organic solvents

The serine proteases alpha-chymotrypsin, trypsin, and subtilisin Carlsberg were immobilized in a sol-gel matrix and the effects on the enzyme activity in organic media are evaluated. The percentage of immobilized enzyme is 90% in the case of alpha-chymotrypsin and the resulting specific enzyme activity in the transesterification of N-acetyl-L-phenylalanine ethyl ester with 1-propanol in cyclohexane is 43 times higher than that of a nonimmobilized lyophilized alpha-chymotrypsin. The activities of trypsin and subtilisin Carlsberg are enhanced with 437 and 31 times, respectively. The effect of immobilization on the enzyme activity is highest in hydrophobic solvents.     

Synthesis of AcPheLeuNH(2) by alpha-chymotrypsin in TTAB reversed micelles: Application of response surface methodology to the optimization of the system

The influence of the long chain alcohols, hexanol, octanol, and decanol, as cosurfactants of the reverse micellar system of tetradecyltrimethylammonium bromide on the alpha-chymotrypsin-mediated AcPheLeuNH(2) synthesis was studied. The effect of temperature, buffer molarity, pH, and substrate concentration was also evaluated. The enzyme was chemically modified and the effect of this modification upon the enzyme activity was also analyzed. Octanol allowed a higher activity for both enzyme forms. The peptide synthesis/substrate hydrolysis ratio is independent of the long chain alcohol used. The chemical modification decreases the alpha-chymotrypsin activity under the system conditions studied, but increases the initial velocity of peptide synthesis relative to the ester substrate hydrolysis. The response surface methodology was applied to optimize the dipeptide synthesis in the system containing octanol as cosurfactant. (c) 1994 John Wiley & Sons, Inc.     

The reactions of alpha-chymotrypsin and related proteins with ester substrates in non-aqueous solvents.

1. The reactivity of alpha-chymotrypsin toward p-nitrophenylacetate has been studied in dimethylformamide, dimethylsulfoxide, formamide and methylacetamide. p-Nitrophenol is liberated in dimethylsulfoxide only. 2. The reactions of alpha-chymotrypsin in dimethylsulfoxide are characterized by the same kinetic and equilibrium constants with either the p-nitrophenyl esters of straight chain carboxylic acids (from acetic to n-caprylic) or with the "specific substrate", N-carbobenzoxy-DL-phenylalanine p-nitrophenyl ester. This signifies that reactions of alpha-chymotrypsin in dimethylsulfoxide, unlike those in aqueous medium, have no specificity toward su-strate structure. 3. The stoichiometry of alpha-chymotrypsin reactions in dimethylsulfoxide was shown to be about five moles of substrate per mole of enzyme. After attaining this stoichiometry, the reaction is completed. 4. Optical rotatory dispersion spectra indicate that in non-aqueous media alpha-chymotrypsin undergoes a large conformational transition which results in a random coil. 5. Chymotrypsinogen, trypsin, trysinogen, lysozyme and serum albumin react with p-nitrophenylacetate in dimethylsulfoxide at rates which are approximately equal to those of alpha-chymotrypsin. Thus, the "activity" of alpha-chymotrypsin in dimethylsulfoxide toward p-nitrophenylacetate does not differ from the "activity" of other proteins, some of which are not even hydrolytic enzymes.     

Evaluation of equilibrium constants for the binding of N-acetyl-L-tryptophan to monomeric and dimeric forms of alpha-chymotrypsin

The binding of N-acetyl-tryptophan to the monomeric and dimeric forms of alpha-chymotrypsin in I = 0.2 acetate-chloride buffer, pH 3.86, has been studied quantitatively. Equilibrium sedimentation studies in the absence of inhibitor yielded a dimerization constant of 3.5 L/g. This value was confirmed by frontal gel chromatography of the enzyme on Bio-Gel P-30, which was also used to establish that N-acetyl-L-tryptophan binds preferentially to monomeric enzyme. From kinetic studies of competitive inhibition with N-acetyl-L-tryptophan ethyl ester as substrate, an equilibrium constant of 1300 M-1 was determined for the binding of N-acetyl-L-tryptophan to monomeric alpha-chymotrypsin. An intrinsic binding constant of 250 M-1 for the corresponding interaction with dimeric enzyme was calculated on the basis of these results and binding data obtained with concentrated (18.5 g/L) alpha-chymotrypsin. The present results refute earlier claims for exclusive binding of competitive inhibitors to monomer and also those for equivalence of inhibitor binding to monomeric and dimeric forms of alpha-chymotrypsin.     

Trypsin-specific acyl-4-guanidinophenyl esters for alpha-chymotrypsin-catalysed reactions computational predictions, hydrolyses, and peptide bond formation.

The function of acyl-4-guanidinophenyl esters as substrate mimetics for the serine protease alpha-chymotrypsin was investigated by protein-ligand docking, hydrolysis, and acyl transfer experiments. On the basis of protein-ligand docking studies, the binding and hydrolysis properties of these artificial substrates were estimated. The predictions of the rational approach were confirmed by steady-state hydrolysis studies on 4-guanidinophenyl esters derived from coded amino acids (which alpha-chymotrypsin is not specific for), noncoded amino acids, and even simple carboxylic acid moieties. Enzymatic peptide syntheses qualify these esters as suitable acyl donors for the coupling of acyl components far from the natural enzyme specificity, thus considerably expanding the synthetic utility of alpha-chymotrypsin.     

Biosensor based on enzyme-catalysed degradation of thin polymer films

A biosensor based on the enzyme-catalysed dissolution of biodegradable polymer films has been developed. Three polymer-enzyme systems were investigated for use in the sensor: a poly(ester amide), which is degraded by the proteolytic enzyme alpha-chymotrypsin; a dextran hydrogel, which is degraded by dextranase; and poly(trimethylene) succinate, which is degraded by a lipase. Dissolution of the polymer films was monitored by Surface Plasmon Resonance (SPR). The rate of degradation was directly related to enzyme concentration for each polymer/enzyme couple. The poly(ester amide)/alpha-chymotrypsin couple proved to be the most sensitive over a concentration range from 4 x 10(-11) to 4 x 10(-7) mol l(-1) of enzyme. The rate of degradation was shown to be independent of the thickness of the poly(ester amide) films. The dextran hydrogel/dextranase couple was less sensitive than the poly(ester amide)/alpha-chymotrypsin couple but showed greater degradation rates at low enzyme concentrations. Enzyme concentrations as low as 2 x 10(-11) mol l(-1) were detected in less than 20 min. Potential fields of application of such a sensor system are the detection of enzyme concentrations and the construction of disposable enzyme based immunosensors, which employ the polymer-degrading enzyme as an enzyme label.     

Detection of native peptides as potent inhibitors of enzymes. Crystal structure of the complex formed between treated bovine alpha-chymotrypsin and an autocatalytically produced fragment, IIe-Val-Asn-Gly-Glu-Glu-Ala-Val-Pro-Gly-Ser-Trp-Pro-Trp, at 2.2 angstroms resolution

Chymotrypsin is a prominent member of the family of serine proteases. The present studies demonstrate the presence of a native fragment containing 14 residues from Ile16 to Trp29 in alpha-chymotrypsin that binds to chymotrypsin at the active site with an exceptionally high affinity of 2.7 +/- 0.3 x 10(-11) M and thus works as a highly potent competitive inhibitor. The commercially available alpha-chymotrypsin was processed through a three phase partitioning system (TPP). The treated enzyme showed considerably enhanced activity. The 14 residue fragment was produced by autodigestion of a TPP-treated alpha-chymotrypsin during a long crystallization process that lasted more than four months. The treated enzyme was purified and kept for crystallization using vapour the diffusion method at 295 K. Twenty milligrams of lyophilized protein were dissolved in 1 mL of 25 mM sodium acetate buffer, pH 4.8. It was equilibrated against the same buffer containing 1.2 M ammonium sulfate. The rectangular crystals of small dimensions of 0.24 x 0.15 x 0.10 mm(3) were obtained. The X-ray intensity data were collected at 2.2 angstroms resolution and the structure was refined to an R-factor of 0.192. An extra electron density was observed at the binding site of alpha-chymotrypsin, which was readily interpreted as a 14 residue fragment of alpha-chymotrypsin corresponding to Ile-Val-Asn-Gly-Glu-Glu-Ala-Val-Pro-Gly-Ser-Trp-Pro-Trp(16-29). The electron density for the eight residues of the C-terminus, i.e. Ala22-Trp29, which were completely buried in the binding cleft of the enzyme, was of excellent quality and all the side chains of these eight residues were clearly modeled into it. However, the remaining six residues from the N-terminus, Ile16-Glu21 were poorly defined although the backbone density was good. There was a continuous electron density at 3.0 sigma between the active site Ser195 Ogamma and the carbonyl carbon atom of Trp29 of the fragment. The final refined coordinates showed a distance of 1.35 angstroms between Ser195 Ogamma and Trp29 C indicating the presence of a covalent linkage between the enzyme and the native fragment. This meant that the enzyme formed an acyl intermediate with the autodigested fragment Ile16-Trp29. In addition to the O-C covalent bond, there were several hydrogen bonds and hydrophobic interactions between the enzyme and the native fragment. The fragment showed a high complementarity with the binding site of alpha-chymotrypsin and the buried part of the fragment matched excellently with the corresponding buried part of Turkey ovomucoid inhibitor of alpha-chymotrypsin.     

Studies on conformation of soluble and immobilized enzymes using differential scanning calorimetry. 2. Specific activity and thermal stability of enzymes bound weakly and strongly to Sepharose CL 4B

Ribonuclease A (EC 3.1.4.22) and alpha-chymotrypsin (EC 3.4.21.1) have been covalently coupled, by a varying number of bonds, to Sepharose CL 4B which was activated with different amounts of CNBr. Upon increasing the number (1-8) of points of attachment between the enzyme and the matrix, the specific activities of immobilized ribonuclease A relative to its soluble counterpart decreased from 60 to 15% while the amount of protein coupled increased from 5 to 37 mg per g of sucked gel. Differential scanning calorimetry was used to determine whether the immobilization caused any changes in the physicochemical properties of the enzyme. Ribonuclease A, weakly bound to the matrix, showed almost the same behavior as the soluble enzyme. By contrast strongly immobilized enzyme exhibited a higher transition temperature (by about 5 degrees C) and a broader endotherm. Similar results were found for alpha-chymotrypsin.     

A simple procedure for the photoregulation of chymotrypsin activity.

A convenient and rapid method for the photo-regulation of the proteolytic enzyme alpha-chymotrypsin is described. When alpha-chymotrypsin is coated with photolytic 1-(2-nitrophenyl)ethanol residues this not only markedly reduces the capability of the enzyme to digest both of the small substrates N-benzoyl-L-tyrosine ethyl ester and N-succinyl-L-phenylalanine p-nitroanilide, but also completely inhibits the enzyme's proteolytic activity. The inactivated alpha-chymotrypsin can then be reactivated under physiological conditions, when and where it is required, by exposure to UV-A light. These results further demonstrate that 1-(2-nitrophenyl)ethanol coated proteins can often be used as light sensitive biological switches as a simple alternative to site directed procedures.     

Fixation of a highly reactive form of alpha-chymotrypsin by micellar matrix

Using reversed micelles of surfactants solvated by water-organic cosolvent mixtures as a matrix for enzyme entrapping, it is possible to fix the highly reactive alpha-chymotrypsin form. The reactivity of alpha-chymotrypsin towards nonspecific substrates increases to the extent comparable with that observed in reactions involving specific substrates.     

Ca2+-independent cyclic GMP phosphodiesterases from rat liver and HTC hepatoma cells.

We have separated and characterized a Ca2+- and calmodulin-insensitive cyclic nucleotide phosphodiesterase from rat liver supernatant as well as an analogous enzyme from HTC hepatoma cells. Chromatography of rat liver supernatant on DEAE-cellulose in the presence and subsequently in the absence of 0.1 mM-CaCl2 resulted in the separation of two distinct phosphodiesterase activities, both of which preferentially hydrolysed cyclic GMP rather than cyclic AMP. One enzyme, E-Ib, was activated in the presence of Ca2+ and calmodulin, and the other, E-Ia, was not. The E-Ia enzyme, which did not bind to calmodulin-Sepharose, had Mr 325 000 and displayed anomalous kinetic behaviour [Km (cyclic GMP) 1.2 microM; Km (cyclic AMP) 15.4 microM]. The E-Ib enzyme, which bound to calmodulin-Sepharose in the presence of Ca2+, had Mr 150 000 and exhibited Michaelis-Menten kinetics for hydrolysis of cyclic GMP [Km (basal) 6.5 microM; Km (activated) 12.0 microM]. E-Ia activity was diminished by incubation with alpha-chymotrypsin and was unaffected by the action of a rat kidney lysosomal proteinase. Partial hydrolysis of E-Ib enzyme by alpha-chymotrypsin or the kidney proteinase resulted in irreversible activation of the enzyme. The E-I enzyme isolated from HTC hepatoma cells was similar to the rat liver E-Ia enzyme in many respects. Its apparent Mr was 325 000. Its activity was unaffected by calmodulin in the presence of Ca2+ or by incubation with the kidney proteinase, and was decreased by digestion with alpha-chymotrypsin. Unlike the liver E-Ia enzyme, however, the hepatoma enzyme exhibited normal kinetic behaviour, with Km (cyclic GMP) 3.2 microM. Although HTC cells contain two other phosphodiesterases analogous to those in rat liver and a calmodulin-like activator of phosphodiesterase, no calmodulin-sensitive phosphodiesterase was detected.     

Raman spectroscopic study of the changes in secondary structure of chymotrypsin: effect of pH and pressure on the salt bridge

Conformational changes of alpha-chymotrypsin, induced by pH and pressure, have been studied with Raman spectroscopy. The secondary structure of alpha-chymotrypsin, chymotrypsinogen and DFP-chymotrypsin has been calculated by a singular value analysis of the Raman amide-I band. The changes in secondary structure, with pH and pressure titration of alpha-chymotrypsin, indicate a conformational transition. The salt bridge between Asp-194 and Ile-16 is disrupted, and the enzyme becomes inactive. No changes are observed for chymotrypsinogen. It is concluded that the proenzyme exhibits the same conformation at different pH values as alpha-chymotrypsin at alkaline pH. The results for DFP-chymotrypsin indicate that the active conformation is stabilized by the presence of the DFP inhibitor in the binding site.     

alpha-Chymotrypsin inhibition studies on the lignans from Vitex negundo Linn

The lignans (1-8) isolated from the roots of Vitex negundo Linn. were screened against the serine proteases alpha-chymotrypsin, thrombin and prolyl endopeptidase. Compounds 3 and 4 were found to be active only against alpha-chymotrypsin and were noncompetitive and competitive inhibitors of the enzyme, respectively. Ki values were found to be in the range 31.75-47.11 microM.     

Interaction of alpha-chymotrypsin with dimethylsulfoxide: a change of substrate could change the mechanism of interaction

The kinetic behavior of alpha-chymotrypsin was studied in water-DMSO mixtures at concentrations of the organic solvent that do not cause irreversible denaturation of the enzyme. Various substrates (N-substituted derivatives of L-tyrosine) were found to display substantially different kinetic patterns of interaction with alpha-chymotrypsin, which can be described by totally different kinetic schemes. The differences were ascribed to competition between the N-acyl group of the substrate and the DMSO molecule at the S2-site of substrate binding to the active site of the enzyme.     

Enzyme-based high-performance liquid chromatography supports as probes of enzyme activity and inhibition: the immobilization of trypsin and alpha-chymotrypsin on an immobilized artificial membrane high-performance liquid chromatography support.

Immobilized artificial membrane (IAM) HPLC supports have been used to immobilize the enzymes alpha-chymotrypsin and trypsin. The enzymes were trapped in hydrophobic cavities on the support and were not covalently attached to the IAM surface. The resulting IAM-enzyme supports retained the hydrolytic activity of the immobilized enzymes: the IAM-trypsin support catalyzed the hydrolysis of N alpha-benzoyl-DL-arginine-p-nitroanilide (BAPNA), and the IAM-alpha-chymotrypsin support (IAM-ACHT) catalyzed the hydrolysis of a number of substrates, including tryptophan methyl ester. The activities of both supports were decreased by known enzyme inhibitors and the activity of the IAM-ACHT was affected by changes in pH and temperature. When a substrate was chromatographed on an IAM-ACHT HPLC, the hydrolytic activity of the immobilized enzyme could be determined from the resulting substrate/product ratios. These data were obtained either directly from the IAM-ACHT chromatogram or from the chromatogram produced by a coupled column system. The results of this study indicate that IAM-immobilized alpha-chymotrypsin and trypsin can be used as chromatographic probes for the qualitative determination of enzyme/substrate and enzyme/inhibitor interactions.     

Properties of alpha-chymotrypsin enclosed into polycarbonate microcapsules. Estimation of the diffusion effect

The conditions for the microencapsulation of alpha-chymotrypsin into semi-permeable polycarbonate membranes are selected. The diameter of the microcapsules is 100-150 microns. The microencapsulation can be carried out at any value of pH. The effect of the diffusion in the hydrolysis of some esters by microcapsulated alpha-chymotrypsin was estimated. The thermostability of the microencapsulated enzyme was studied.     

Interaction of methylchymotrypsin with Streptomyces subtilisin inhibitor

The effect of methylation of histidine-57 of alpha-chymotrypsin with Streptomyces subtilisin inhibitor was examined. Methylchymotrypsin was isolated by affinity chromatography on inhibitor-Sepharose, and the interaction of this inactive enzyme with inhibitor was quantitatively analyzed by two different methods: the spectrophotometric titration of difference spectrum resulted in the complex formation and the application of competitive enzyme assay by using substrates of large Km values. The former method gave values of 8.6 . 10(-6) M as dissociation constant (Kd) of methylchymotrypsin . inhibitor complex and 0.91 as the number of binding sites (n) per inhibitor monomer, both of which were almost equivalent to those for native enzyme . inhibitor complex. By the latter novel method, values of 7.9 . 10(-6) M and 1.08 were obtained for Kd and n, respectively, for interaction of inhibitor with alpha-chymotrypsin, and 8 . 10(-6) M as Kd for methylchymotrypsin . inhibitor complex. These results indicate that methylation of histidine-57 of active site in alpha-chymotrypsin molecule does not affect essentially the binding ability to inhibitor and the modified enzyme binds stoichiometrically to inhibitor, as the native enzyme does, with a molar ratio of 1:1 per inhibitor monomer.     

Artificial oligomerization of enzymes--a new way of regulating their catalytic activity in reversed micellar systems]

Comparative studies were carried out in the catalytic activity regulation of native alpha-chymotrypsin and its artificially produced hexameric form as an example of non-dissociating oligomeric enzyme (covalently cross-linked by means of succinimidyl-3-(2-pyridylthiopropionate] in the Aerosol OT reversed micelles in octane. Native (monomeric) alpha-chymotrypsin exhibits maximal catalytic activity in the reversed micelles at the hydration degree w0 = 10, when the radius of the micelle inner cavity is equal to the radius of the alpha-chymotrypsin globule. For the alpha-chymotrypsin hexamer, optimum is observed at w0 = 45, with the inner micellar cavity radius (r = 68 A) being approximately equal to the radius of the sphere surrounding the octahedral combination of the six monomeric alpha-chymotrypsin molecules (r = 61 A). Thus, construction of the corresponding oligomeric structures is made easy, with the optimal catalytic activity in a preset range of the hydration degrees.