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 binding of the fluorescence probe 1-anilinonaphthalene-8-sulfonate (Ans) to alpha-chymotrypsin (alpha-CHT) at pH 3.6 is accompanied by a dramatic enhancement of Ans fluorescence and a shift of the emission maximum to shorter wavelengths. Our study reveals that one Ans molecule binds to alpha-CHT at a site different from either the active site of alpha-CHT or the 2-p-toluidinylnapthalene-6-sulfonate binding site. the binding constant of Ans is about the same (10(4) M-1) at pH 3.6 and 6.4. Nanosecond fluorescence depolarization data indicate that Ans is rigidly bound to alpha-CHT. The fluorescence enhancement due to binding of Ans to alpha-CHT at low pH could be due to binding either to a hydrophobic site or to a site where local dipoles do not relax during the excited-state lifetime of Ans. As the pH is increased, fluorescence intensity of the Ans-alpha-CHT complex decreases appreciably; and the emission maximum shifts to longer wavelengths. The fluorescence decay curves exhibit a corresponding sensitivity to pH. The pH effect on the fluorescence of Ans-alpha-CHT can be interpreted in terms of a pH-dependent equilibrium between alpha-CHT conformers differing in the degree of mobility of polar residues and water molecules at the Ans binding site or structural changes in the Ans binding site.
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PMID:Interaction of alpha-chymotrypsin with the fluorescent probe 1-anilinonaphthalene-8-sulfonate in solution. 42 15

The rates of hydrolysis of p-nitrophenyl acetate (pNPA), p-nitrophenyl propionate (pNPP), p-nitrophenyl butanate (pNPB), and p-nitrophenyl valerate (pNPV) catalyzed by alpha-chymotrypsin (alpha-CHT) were measured with and without volatile anesthetics at 25.0 degrees C. Halothane activated the hydrolysis of pNPA and pNPP, meanwhile inhibited that of pNPB and pNPV. The activation phenomena were explained by the existence of a 1:1 enzyme-anesthetics complex and the opening of an activated pathway. The rate constant of pNPA hydrolysis catalyzed by alpha-CHT of the activated pathway kA by halothane was 0.269 s-1, whereas that of the normal pathway was k0 0.093 s-1. The free energy of activation was stabilized at 0.64 kcal/mol by halothane. The mechanisms of the activation and inhibition are discussed in terms of the molecular size of the substrate and anesthetics.
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PMID:Volatile anesthetics-induced activation phenomena of alpha-chymotrypsin-catalyzed hydrolysis. 158 46

Crystals of gamma-chymotrypsin (gamma-CHT) grown at pH 7.0 are stable from pH 2.0 to 11.0. Crystalline gamma-CHT therefore provides an unusually favourable system to observe the structure of a protein and its bound solvent over a broad range of pH. In this report we describe the high-resolution refined structure of gamma-CHT at pH values of 2.0, 7.0 and 10.5. The apparent tetrapeptide seen bound in the active site of gamma-CHT at pH 7.0 is also present at pH 2.0 and 10.5 although it is better defined at low pH. A comparison of the respective structures shows that there is additional electron density in the low pH structure at the point where the side-chain of Ser 195 approaches most closely to the presumptive inhibitor. This suggests that the adduct is most likely to be covalently linked to the enzyme at low pH and to be non-covalent at higher pH. As the pH is lowered from 7.0 to 2.0, the side-chain of His 40 rotates approximately 120 degrees about its C alpha-C beta bond and, in concert, the side-chain of Gln 34 also rotates approximately 140 degrees about its C alpha-C beta bond. Apart from these localized rearrangements in the vicinity of His 40, the structure of gamma-CHT at pH 2.0 is very similar to that at neutral pH. The structure of gamma-CHT at pH 10.5 is also seen to be almost identical with that at neutral pH. There is no indication that the internal salt bridge between Asp 194 and the alpha-amino group of lle 16 begins to dissociate at pH 10.5. With the exception of the vicinity of His 40, the structure of the bound solvent in the crystal structures at low, neutral and high pH is very similar.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Structure of gamma-chymotrypsin in the range pH 2.0 to pH 10.5 suggests that gamma-chymotrypsin is a covalent acyl-enzyme adduct at low pH. 188 17

A 1.8-A resolution x-ray crystallographic restrained least squares refinement has been carried out on the phenylethane boronic acid (PEBA) complex of alpha-chymotrypsin dimer (alpha-CHT), and it has been compared to the 1.67-A resolution structure of the native enzyme. PEBA has a high binding affinity for alpha-CHT, and the boronate forms a tetrahedral complex with Ser-195 OG of one molecule of the dimer; the boronate in the other molecule is severely disordered and does not form a tetrahedral complex. The former could be a model of the transition state of catalysis. The complex of PEBA X alpha-CHT displays significant nonequivalence in conformation of side chains between the independent molecules comparable to the native enzyme, but, like the latter, shows a high degree of fidelity in the folding of the main chain. The orientation of the phenyl ring, CA and CB of PEBA, in the specificity sites of the two molecules is similar, suggesting that recognition is fairly insensitive to small departures from local symmetry; the same does not apply to the boronate functionalities suggesting that greater precision is required for catalysis. The folding of the molecule remains the same upon PEBA binding, but some of the side chains respond nonequivalently. The latter is a consequence of the inherent nonequivalence of the native dimer and the asymmetrical nature of the PEBA binding.
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PMID:Structure of a tetrahedral transition state complex of alpha-chymotrypsin dimer at 1.8-A resolution. 358 39

The crystal structure of bovine alpha-chymotrypsin (alpha-CHT) in complex with the bovine basic pancreatic trypsin inhibitor (BPTI) has been solved and refined at 2.8 A resolution (R-factor = 0.18). The proteinase:inhibitor complex forms a compact dimer (two alpha-CHT and two BPTI molecules), which may be stabilized by surface-bound sulphate ions, in the crystalline state. Each BPTI molecule, at opposite ends, is contacting both proteinase molecules in the dimer, through the reactive site loop and through residues next to the inhibitor's C-terminal region. Specific recognition between alpha-CHT and BPTI occurs at the (re)active site interface according to structural rules inferred from the analysis of homologous serine proteinase:inhibitor complexes. Lys15, the P1 residue of BPTI, however, does not occupy the alpha-CHT S1 specificity pocket, being hydrogen bonded to backbone atoms of the enzyme surface residues Gly216 and Ser217.
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PMID:Crystal structure of the bovine alpha-chymotrypsin:Kunitz inhibitor complex. An example of multiple protein:protein recognition sites. 917 77

The anticoagulant human plasma serine protease, activated protein C (APC), inactivates blood coagulation factors Va (FVa) and VIIIa. The so-called autolysis loop of APC (residues 301-316, equivalent to chymotrypsin [CHT] residues 142-153) has been hypothesized to bind FVa. In this study, site-directed mutagenesis was used to probe the role of the charged residues in this loop in interactions between APC and FVa. Residues Arg306 (147 CHT), Glu307, Lys308, Glu309, Lys311, Arg312, and Arg314 were each individually, or in selected combinations, mutated to Ala. The purified recombinant protein C mutants were characterized using activated partial thromboplastin time (APTT) clotting assays and FVa inactivation assays. Mutants 306A, 308A, 311A, 312A, and 314A had mildly reduced anticoagulant activity. Based on FVa inactivation assays and APTT assays using purified Gln506-FVa and plasma containing Gln506-FV, it appeared that these mutants were primarily impaired for cleavage of FVa at Arg506. Studies of the quadruple APC mutant (306A, 311A, 312A, and 314A) suggested that the autolysis loop provides for up to 15-fold discrimination of the Arg506 cleavage site relative to the Arg306 cleavage site. This study shows that the loop on APC of residues 306 to 314 defines an FVa binding site and accounts for much of the difference in cleavage rates at the 2 major cleavage sites in FVa. (Blood. 2000;96:585-593)
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PMID:The autolysis loop of activated protein C interacts with factor Va and differentiates between the Arg506 and Arg306 cleavage sites. 1088 22