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 15 exposed carboxyl groups of alpha-chymotrypsin were modified with glycine ethyl ester at low pH using barbodiimide reagent. The specificity of the modified enzyme (Chy-15) was studied over the pH range of 4 to 9 with both N-acylated and non-N-acylated amino acid esters. The modified enzyme had lower reactivity toward N-acylated esters than non-N-acylated esters compared to the native enzyme. Typical substances such as acetyl- and benzoyl-L-tyrosine ethyl esters retained 4 and 9% activity, whereas phenylalanine ethyl ester was slightly more reactive with the modified than with the native enzyme. The pH-rate profiles of acetyl-L-phenylalanine ethyl ester and tryptophan ethyl and benzyl esters were investigated in detail. Analysis of these profiles revealed three pKa values of approximately 5, 7, and 9 related to a functional carboxyl, imidazoyl, and an amino group, respectively. Since similar pKa values occur for the native enzyme, modification did not block the carboxyl corresponding to pKa 5. A mechanism is proposed for catalysis which includes both the protonated and unprotonated form of the imidazoyl (His-57) and utilizes water rather than a carboxyl (Asp-102) as the proton sink.
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PMID:Specificity of alpha-chymotrypsin with exposed carboxyl groups blocked. 0 45

Two proteinases (2A and 2B) purified from the granular fraction of horse blood leucocytes degrade casein (Km values 12.8 and 6mg/ml respectively) with maximum activity at pH 7.4 and in the presence of 2m-urea. Urea-denatured haemoglobin, fibrinogen, albumin and resorcin/fuchsin-stained elastin are digested at a slower rate. The enzymes hydrolyse synthetic substrates of elastase, N-benzyloxycarbonyl-L-alanine 4-nitrophenyl ester (Km 0.114 and 0.178 mM) and N-acetyl-tri-L-alanine methyl ester (Km 5.55 and 0.98 mM), but they do not hydrolyse synthetic substrates of trypsin, chymotrypsin and thrombin. The examined proteinases are completely inhibited by 2 mM-di-isopropyl phosphorfluoridate and show a sensitivity to butyl and octyl isocyanates similar to that of pancreatic elastase. The pH-dependence of their photoinactivation in the presence of Rose Bengal indicates the presence of histidine in the active centre. Proteinase 2A rather insensitive to iodination by IC1 as is pancreatic elastase, whereas proteinase 2B is totally inactivated after incorporation of five iodine atoms per enzyme molecule.
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PMID:Substrate specificity and modifications of the active centre of elastase-like neutral proteinases from horse blood leucocytes. 0 9

When the competitive inhibitor benzeneboronic acid (BBA) forms a complex with alpha-chymotrypsin [EC 3.4.21.1] protons are released in the acidic pH region. The proton release can be measured by a difference potentiometric technique. The proton release is also observed in chymotrypsinogen A but not in TRCK-, DIP-, and anhydrochymotrypsins. Based on these observations, a simple procedure to estimate the equilibrium constants of the trigonal-tetrahedral interconversion of BBA is proposed. Thermodynamic parameters of the ionization of His 57 and of each step involved in BBA binding can be estimated from the temperature dependence of the proton release. Those of His 57 are essentially the same as those of imidazole in water. Regarding the interconversion of BBA on the enzyme, the value of delta S is similar to delta S not equal to of the deacylation step of nonspecific substrates, and delta H is remarkably reduced from that for the ionization of BBA in water. The enthalpic gain of enzymic process is suggested to be due to the change of the proton acceptor, which is water in the case of the ionization of BBA in water, to imidazole on the enzyme.
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PMID:Potentiometric studies on benzeneboronic acid-alpha-chymotrypsin interactions. 0 50

The modification of alpha-chymotrysin with phenacyl bromide has been reinvestigated over a wide pH range. Evidence is presented that indicates that the nature of the phenacyl-modified enzymes prepared by this reaction is dependent upon the pH of the reaction medium. The phenacyl alpha-chymotrypsin produced at low pH is most probably the Met-192 phenacylsulfonium salt, as proposed earlier, since it readily undergoes dealkylation using 2-mercaptoethanol. However, the phenacyl-enzyme prepared at neutral pH possesses a much reduced enzymatic activity and does not react with 2-mercaptoethanol to regenerate native alpha-chymotrypsin. In addition, incubation of the Met-192 phenacyl sulfonium enzyme at neutral pH causes a smooth irreversible change to the new phenacyl-enzyme as monitored by changes in enzymatic activity, susceptibility to dealkylation using 2-mercaptoethanol, and ultraviolet difference absorption spectral properties. The stoichiometries of both the low and neutral pH modification reactions have been determined, using [carbonyl-14C]phyenacyl bromide, to be 1 phenacyl group/enzyme molecule. In efforts to obtain information about the nature and mechanism of formation of the phenacyl alpha-chymotrypsin produced at neutral pH, alkylation reactions of modified alpha-chymotrypsins produced by His-57 functionalization with tosylphenylalanine chloromethyl ketone and by Met-192 oxidation to the sulfoxide have been investigated. The combined results of these studies have been initially interpreted in terms of a neutral pH, phenacyl bromide modification resulting in formation of a new modified enzyme via the Met-192 sulfonium salt.
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PMID:Reinvestigation of the phenacyl bromide modification of alpha-chymotrypsin. 0 93

The interaction of chymotrypsinogen A with benzeneboronic acid (BBA), a transition state along inhibitor of serine proteases, was investigated by the temperature-jump method using pH indicators. It was found that l/tau is dependent on BBA concentration, in contrast to the case of the alpha-chymotrypsin [EC 3.4.21.1]-BBA system in which l/tau is independent of BBA concentration. By examination of the pH dependences of the kinetic parameters, the acid dissociation behavior of His 57 in chymotrypsinogen, chymotrypsinogen-trigonal BBA complex and chymotrypsinogen-tetrahedral BBA complex was analyzed. The kinetic deuterium isotope effect was also examined and found to occur principally on the acid dissociation constants. The state of the catalytic residues in the zymogen molecule is discussed based on these results.
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PMID:Temperature-jump studies on the interaction of benzeneboronic acid with chymotrypsinogen. 1 Feb 89

The reactions between yeast carboxypeptidase C and the group-specific reagents, phenylglyoxal and iodoacetamide, have been studied in detail and the reactions of residue at the active site with N-tosyl-L-phenylalanine chloromethyl ketone and diisopropyl phosphorofluoridate have been confirmed. Modification of the enzyme by either phenylglyoxal or iodoacetamide results in the loss of peptidase activity, while esterase activity remains unchanged. Inactivation by phenylglyoxal appears to be the result of the modification of a single arginine residue, whereas inhibition by iodoacetamide can be correlated with the modification of a single methionine residue. Inactivation of the enzyme by either N-tosyl-L-phenylalanine chloromethyl ketone or diisopropyl phosphorofluoridate is the result of the modification of a single histidine and a single serine residue, respectively. The pattern of inhibition indicates certain analogies in the mechanism of yeast carboxypeptidase C to pancreatic chymotrypsin, on the one hand, and to carboxypeptidase A, on the other.
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PMID:Reaction of yeast carboxypeptidase C1 with group-specific reagents. 1 Sep 62

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

A detailed examination of the mechanism of the hydrolysis of phenyl acetates by alpha-chymotrypsin [EC 3.4.21.1] was carried out. The effective deacylation rate constants of some phenyl acetates obtained by titration of the acetyl-enzyme decreased at low substrate concentrations and showed anomalous pH dependences and solvent isotope effects. The transient kinetics of deacylation of the acetyl-enzyme were biphasic. A spectrum and a breakdown rate similar to those of acetylimidazole were observed when the acetyl-enzyme was denaturated with sodium dodecyl sulfate. These results indicate the participation of histidine-acylated enzyme, which woud account for the anomalous phenomena previously found in this system, including a large value of Hammett's rho. The relation between the substrate activation and the two intermediates is discussed.
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PMID:alpha-chymotrypsin-catalyzed hydrolysis of phenyl acetate. Large Hammett's rho constant and participation of histidine-acylated intermediate. 1 53

Three new protease inhibitors were isolated and purified about 200-fold from hemolymph of silkworm larvae, Bombyx mori, using ion-exchange and affinity chromatography. Two of the three inhibitors were basic proteins (SCI-I had pI 9.4 and SCI-II had pI 9.6) and one was acidic (SCI-III had pI 4.0). The molecular weight of each inhibitor was determined to be 7,000 by the sedimentation equilibrium method. The amino acid composition of the inhibitors were similar except for the contents of Asp, Glu, Ile, Leu, and Lys. Val, His, and Trp were not present in the inhibitors and Met appeared only in SCI-III. The CD spectra of the inhibitors were all similar and indicated a low content of alpha-helical structure (10% at most). Each inhibitor could inhibit the protease and esterase activities of bovine alpha-chymotrypsin at a one-to-one molar ratio, and the dissociation constants were 3.1 X 10(-9)M for SCI-I and II and 1.3 X 10(-8)M for SCI-III. Only SCI-II showed a weak inhibitory activity against bovine trypsin. Subtilisin BPN' and papain were not inhibited by these inhibitors.
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PMID:Chymotrypsin inhibitors from hemolymph of the silkworm, Bombyx mori. 2 86

The mechanism of the acid dimerization of alpha-chymotrypsin in solution was reexamined using a number of chemical derivatives. Blocking of the carboxyl of Tyr-146, while that of ASP-64 remained free, eliminated completely the ability of alpha-chymotrypsin to dimerize, as did methylation of His-57. O-Acetylation of Tyr-146 reduced the dimerization constant to that of gamma-chymotrypsin, and deacetylation of the other accessible tyrosines did not affect the dimerization. It is concluded that the mechanism proposed by Aune and Timasheff [Aune, K.C., and Timasheff, S.N.(1971) Biochemistry 10, 1609-1617] for the solution dimerization which involves the electrostatic interaction between the His-57 imidazolium ring and the terminal carboxyl of Tyr-146 is still most consistent with all the experimental observations. The interactions in dilute solution may differ somewhat from those observed in crystals. In particular, the two intermolecular bridges formed by sulfate ions in crystals cannot be present in solution.
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PMID:Low pH dimerization of chymotrypsin in solution. 3 Apr 71


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