Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: 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)

Inferences about the catalytic mechanism of acetylcholinesterase (acetylcholine hydrolase, EC 3.1.1.7) are frequently made on the basis of a presumed analogy with chymotrypsin, EC 3.4.21.1. Although both enzymes are serine hydrolases, several differences in the steady-state kinetic properties of the two have been observed. In this report particular attention is focused on the second-order reaction constant, kcat/Kapp. While the reported pH dependence and deuterium oxide isotope effect associated with this parameter for chymotrypsin are generally consistent with simple models involving rate-limiting general acid-base catalysis, this study finds a more complicated situation with acetylcholinesterase. The apparent pKa of kcat/Kapp for acetylcholinesterase varies between 5.5 and 6.3 for neutral substrates and involves nonlinear inhibition by [H+]. Deuterium oxide isotope effects for kcat/Kapp range from 1.1 for acetylcholine to 1.9 for p-nitrophenyl acetate. The bimolecular reaction rate appears rate-limiting for acetylcholine at low concentrations, while a rate-limiting induced-fit step is proposed to account for apparent pKa values and low deuterium oxide isotope effects associated with low concentrations of phenyl acetate and isoamyl acetate.
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PMID:Catalysis by acetylcholinesterase: evidence that the rate-limiting step for acylation with certain substrates precedes general acid-base catalysis. 0 Jun 68

From a crude extract of chick peas (Cicer arietinum L.) inhibitors of trypsin and chymotrypsin were isolated by affinity chromatography on a column of trypsin-Sepharose 6B. The content of inhibitors was found to be 1.5 g/kg. They were further separated into six isoinhibitors by ion-exchange chromatography on DEAE-Sephadex A-25. Two of the isoinhibitors accounted for about 50% of the isolated inhibitors and were further purified to a homogeneous state. The isoinhibitors had a molecular weight of about 10000 as determined by molecular-sieve chromatography on Sephadex G-75. They were stable towards extremes of pH and temperatures up to 75 degrees C or towards digestion by pepsin. They were also stable in 6 M urea but not in 6 M guanidine-HCl. The intact inhibitors were destroyed when the peas were cooked at 100 degrees C or when they were toasted at 130 degrees C. The four major inhibitors had similar amino acid compositions and did not contain detectable amounts of free sulfhydryl groups, tryptophan or carbohydrate. Cysteine is the dominant amino acid residue in all of them and accounted for about 20% of their amino acid content. The isoelectric point of the isoinhibitors lies in the range of pH 4.9-8.6 and two of the major inhibitors had isoelectric points of pH 4.75 and pH 4.96. They inhibited chymotrypsin to the same extent but differed in their inhibitory activities towards trypsin, indicating that they are mixtures of native and trypsinmodified forms and that they probably have separate sites for the two enzymes. They did not inhibit other proteolytic enzymes belonging to two groups (i.e., serine or cysteine enzymes) or originating from different sources (i.e., animals, plants or bacteria).
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PMID:The trypsin and chymotrypsin inhibitors in chick peas (Cicer arietinum L.). Purification and properties of the inhibitors. 0 Dec 66

Two proteinase inhibitors, designated as inhibitors I and II, were purified from adzuki beans (Phaseolus angularis) by chromatographies on DEAE- and CM-cellulose, and gel filtration on a Sephadex G-100 column. Each inhibitor shows unique inhibitory activities. Inhibitor I was a powerful inhibitor of trypsin [EC 3.4.21.4], but essentially not of chymotrypsin ]EC 3.4.21.1]. On the other hand, inhibitor II inhibited chymotrypsin more strongly than trypsin. The molecular weights estimated from the enzyme inhibition were 3,750 and 9,700 for inhibitors I and II, respectively, assuming that the inhibitions were stoichiometric and in 1 : 1 molar ratio. The amino acid compositions of both inhibitors closely resemble those of low molecular weight inhibitors of other leguminous seeds: they contain large amounts of half-cystine, aspartic acid and serine, and little or no hydrophobic and aromatic amino acids. Inhibitor I lacks both tyrosine and tryptophan residues. The molecular weights were calculated to be 7,894 and 8,620 for inhibitors I and II, respectively. The reliability of these molecular weights was confirmed by the sedimentation equilibrium and 6 M guanidine gel filtration methods. On comparison with the values obtained from enzyme inhibition, it was concluded that inhibitor I and two trypsin inhibitory sites on the molecule, whereas inhibitor II had one chymotrypsin and one trypsin inhibitory sites on the molecule.
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PMID:Purification and characterization of proteinase inhibitors from adzuki beans (Phaseolus angularis). 0 91

The chemical modification of two new double-headed-protease inhibitors from black-eyed peas, a trypsin-chymotrypsin inhibitor (BEPCI) and a trypsin inhibitor (BEPTI) with dansyl chloride was investigated under various conditions. The NH2-terminal serine of both BEPCI and BEPTI, the 4 lysyl residues of BEPCI, and 4 of the 5 lysyl residues of BEPTI, could not be dansylated in the absence of urea. The single tyrosine per subunit of BEPCI and BEPTI was unreactive even in the presence of urea but could be labeled with half-site reactivity by the Celite method. Lysine, NH2-terminal serine, and tyrosine were reactive in fully reduced, carbamidomethylated BEPCI and BEPTI. Gel filtration was used to study the subunit interactions of BEPCI and BEPTI. At pH 8 or pH 3.0 there is a complex set of multiple equilibria with widely differing rates of attainment. We have found evidence for a rapid dimer-tetramer equilibrium, a distinct moderate rate dimer-tetramer equilibrium, a very slow monomer-dimer equilibrium, and postulate slow isomerization of the two forms of dimer and the two forms of tetramer. The monomer-dimer equilibrium is quite unusual in that the dimer is stabilized by chaotropic ions and even slightly by guanidine HC1. In contrast to the complex pattern seen in native BEPCI, the half-site, dansylated BEPCI exists at similar concentration exclusively as a tetramer at neutral pH.
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PMID:Double-headed protease inhibitors from black-eyed peas. III. Subunit interactions of the native and half-site chemically modified proteins. 0 94

The metal content of carboxypeptidase Y was analyzed by the atomic absorption method. After exhaustive dialysis against an EDTA solution, the enzyme showed no loss of activity nor any significant content of metals (Zh,Mg,Ca,Cu,Mn,Ni,Fe, and Co). The activity was, however, rather sensitive to preincubation with various metals. The reactivity of a serine residue of the enzyme was also reevaluated. Diisopropyl fluorophosphate (DFP) and phenylmethanesulfonyl fluoride (PMSF) stoichiometrically and irreversively inhibited the enzyme. The rate of inactivation with DFP was much faster than that for typsin [EC 3.4.21.4] and chymotrypsin [EC 3.4.21.1.], while the rate with PMSF was one-fifteenth of that for chymotrypsin. The pH-dependence of the inactivation by DFP was similar to that of the enzymatic hydrolysis of acetylphenylalanine ethyl ester. The present results indicate that carboxypeptidase Y is free of metals and has a serine residue with a vital role in the catalytic process, though the functional role of this SH group remains to be clarified.
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PMID:Further confirmation of carboxypeptidase Y as a metal-free enzyme having a reactive serine residue. 0 4

1. The specificity of cathepsin G, a neutral proteinase from human spleen, was examined by use of low-molecular-weight substrates. The enzyme was found to hydrolyse several synthetic substrates also hydrolysed by chymotrypsin, but with different kinetic constants. 2. Maximal activity against benzoyl-DL-phenylalanine 2-naphthol ester and azo-casein was in the range pH 7.5-8.0. 3. The sensitivity of cathepsin G to the action of potential inhibitors was determined, and compared with those of bovine chymotrypsin and subtilisin. Cathepsin G showed the characteristics of a serine proteinase, but was less affected by the chloromethyl ketone of tosylphenylalanine than was chymotrypsin. 4. A rabbit anti-(human cathepsin G) serum was raised, and precipitin lines formed in agarose gel were stained for activity of the enzyme. 5. Cathepsin G was shown to be immunologically identical with the chymotrypsin-like enzyme of the azurophil granules of the neutrophil granulocytes.
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PMID:Human cathepsin G. Catalytic and immunological properties. 0 45

5-Dimethylaminonaphthalene-1-sulfonyl fluoride was evaluated as a reagent for the selective labeling of proteins. In a comparative study with Dns-chloride a greatly increased selectivity of the fluoride was found with a number of proteins. The reaction of Dns-fluoride with alpha-chymotrypsin, subtilisin Carlsberg and trypsin was found to be highly specific, resulting in a stoichiometric incorporation of the Dns label with concomitant loss of enzymatic activity. The reaction of Dns-chloride with the same proteinases is unspecific. Evidence was obtained to indicate that reaction of the serine esterases with Dns-fluoride occurs exclusively at the active serine residue. The stability of Dns-fluoride labeled chymotrypsin was investigated. The conjugate was found to be fairly stable in the pH range from 3 to 9 at 25 degrees C and is therefore suitable for fluorescence investigations of the chymotrypsin active-site. Molar extinction coefficients for Dns-labeled serine proteinases were determined using radiocative label.
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PMID:Specific fluorescent derivatives of macromolecules. Reaction of dansyl fluoride with serine proteinases. 0 13

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


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