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 standard free energy (deltaG degrees), enthalpy (deltaH degrees), and entropy (deltaS degrees) of association for proflavin and D- and L-N-AcTrp have been obtained at pH 7.8 for native alpha-chymotrypsin (Cht) and for forms of Cht in which essential catalytic residues of the active site are modified. The modified Cht forms studied are dehydroalaninyl-195-alpha-Cht and N-methylhistidinyl-57-alpha-Cht. Associations to native Cht (pH 7.8) are characterized by negative deltaH degrees and deltaS degrees values (i.e., for L-AcTrp deltaH degrees = -9.1 kcal/mol and deltaS degrees = -21 eu at T = 25 degreesC). In contrast, we found associations to modified Chts to be characterized by an enthalpy near zero and a positive entropy of association, the values of the deltaH degrees and deltaS degrees for association to the modified Cht forms being similar to those expected for transfer of small aromatic molecules from water to a nonpolar solvent phase. Differences in deltaH degrees and deltaS degrees observed for binding of substrate analogues and inhibitors to modified and native Cht (pH 7.8) are approximately + 10 kcal/mol and +30 eu, respectively. Data from D. D. F. Shiao ((1970), Biochemistry 9, 1083) similarly show differences of comparable magnitude between binding of substrate analogues to active alpha-Cht (pH 7.8) and the His-57 protonated form of alpha-Cht (pH 5.6). The negative deltaH degrees and deltaS degrees values of associations for binding to active alpha-Cht indicate that a substrate-induced conformational change occurs on substrate association with the primary binding site (S1), which does not occur in Ser-195 and His-57 modified Cht. From these differences we infer a linkage between binding of substrate into S1 and the catalytic residues in the nucleophilic subsite (S1-S1'). Our data also show that associations of substrate analogues into potentially productive Michaelis complexes S1 cannot be easily differentiated from associations that are nonproductive (i.e., nonactivated) from their deltaG degrees obsd, but may be differentiated by their respective deltaH degrees obsd and deltaS degrees obsd for association. Accordingly, it is indicated that the probable substrate association-activation process, characterized thermodynamically in this work, occurs in the substrate binding step and leads to lowered free energies of activation in catalytic steps succeeding binding however, the process does not influence the observed strength of substrate binding.
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PMID:Thermodynamics of binding to native alpha-chymotrypsin and to forms of alpha-chymotrypsin in which catalytically essential residues are modified; a study of "productive" and "nonproductive" associations. 86 Dec 5

Water-soluble poly(N-vinylpyrrolidone) of molecular weight 10 000 was modified by hydrolysis of 5% of the gamma-lactam rings, and formation of the N-hydroxysuccinimidester. This activated polymer was bound covalently to trypsin (EC 3.4.21.4) and chymotrypsin (EC 3.4.21.1) to complexes of a molecular weight of about 150 000. The bound enzymes showed an increase in stability towards autolysis. Towards small molecular weight substrates the trypsin-poly (N-vinylpyrrolidone) conjugate showed an increase in specific activity of 1: 1.6, whereas the activity towards larger substrate was found to be only 20%. Chymotrypsin-poly(N-vinylpyrrolidone) showed decreased activity against small (50%) and large (7%) molecular weight substrates.
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PMID:Preparation and properties of trypsin and chymotrysin coupled covalently to poly (N-vinylpyrrolidone). 88 41

The 5-dimethylaminonaphthalene-1-sulfonyl group was specifically introduced into the active site region of the serine proteinases: alpha-chymotrypsin, trypsin and subtilisin Carlsberg by the method of affinity-labeling. The resulting fluorescent derivatives were studied by a variety of fluorescence techniques and the results were correlated with structural data available on these enzymes from X-ray analysis. As model compounds for the Dns-proteinases, the absorption and fluorescence properties of Dns-amide and Dns-ethyl ester were studied in ethanol/water and p-dioxane/water mixtures. The fluorescence emission transtion energies and quantum yields were related to four commonly employed solvent-polarity scales. Best correlations for different solvents were obatined with the empirical "Z" and "Y" scales. From inspection of the fluorescence emission transition energies of the Dns group in the Dns-proteinases and comparision with the model compound studies it was possible to assign "Z" values for the apparent microenvironment polarities of the Dns group in the Dns-proteinases. The apparent polarities of the microenvironments of the Dns group in Dns-Ser 195-chymotrypsin (Dns-chymotrypsin (I)); (Dns-Phe-CH2)-His 57-chymotrypsin; (Dns-Lys-CH2)-His 46-trypsin; and Dns-Ser 221--subtilisin Carlsberg (Dns-subtilisin (I)) are in the range of 89.5-92.5 on the "Z" scale. The apparent microenvironment polarity of the Dns group in Dns-Ser 183-trypsin (Dns-trypsin (I)) appears to be below 76.7 on the "Z" scale. The Dns group in Dns-chymotrypsin (I) and (Dns-Phe-CH2)-His 57-chymotrypsin appears to be rigidly bound as evaluated by fluorescence polarization studies. The effect of 2H2O on the fluorescence emission quantum yields of Dns-amide and Dns-ethyl ester was examined. In both cases the ratios of quantum yields in 2H2O:ethanol (8:2) to quantum yields in H2O:ethanol (8:2) was about 1.8. The 2H2O effect upon the fluorescence emission quantum yields of the Dns group has been used to investigate solvent accessibility of this chromophore in the Dns-proteinases. Acessibility studies using 2H2O are very promising and have some definite advantages over other existing methods. Energy transfer between the Trp residues and the bound Dns group was investigated in the Dns-proteinases. The mean transfer distance calculated from the observed transfer efficiencies are 18.1 A, 19.7 A and 18.4 A for (Dns-Phe-CH2)-His 57-chymotrypsin, Dns-chymotrypsin (I) and (Dns-Lys-CH2)-His 46-trypsin, respectivly. From models built using X-ray crystallographic coordinates for the protein atoms, the mean distance of separation between the Trp residues and the bound Dns group for the same set of conjugates ar 18.6 A, 17.5 A and 17.5 A, respectively. Considering the inherent difficulties in energy transfer studies, the results are in excellent agreement with the X-ray data.
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PMID:Specific fluorescent derivatives of macromolecules. A fluorescence study of some specifically modified derivatives of chymotrypsin, trypsin and subtilisin. 95 53

These studies describe the production of murine migration inhibitory factor (MIF)3 in sufficient quantities to allow its partial characterization by physiochemical and enzymatic methods. MIF was obtained from murine spleen cell cultures (C57BL/6 strain) stimulated with concanavalin A (Con A). Characterization of murine MIF was performed using Sephadex G-100 gel chromatography, isopycnic centrifugation in a CsCl density gradient, polyacrylamide disc electrophoresis, heat stability, and enzymatic treatment. MIF-containing and control fractions were assayed on normal C57BL/6 peritoneal exudate cells by using a microcapillary tube assay. Peak MIF activity was found in a Sephadex G-100 fraction containing molecules the size of albumin and slightly smaller, molecular weight 67,000 to 48,000. Murine MIF was stable to heating at 56 degrees C for 30 min but lost its activity at 80 degrees C for 30 min. Incubation of G-100 fractions containing MIF with water insoluble chymotrypsin destroyed the activity of MIF, indicating its protein nature. CsCl density gradient centrifugation revealed that murine MIF had a buoyand density greater than protein, consistent with its being a glycoprotein. Further, when subjected to disc electrophoresis on polyacylamide gels, murine MIF migrated in a region cathodal to albumin. Thus, mitogen stimulation of murine spleen cells produced MIF in quantities which allowed its partial characterization and purification, and its comparison with human and guinea pig MIF; this makes it feasible to analyze the role of murine MIF in cellular immunity and in its relationship to lymphocyte mediators which regulate humoral immune responses.
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PMID:Partial characterization of murine migration inhibitory factor (MIF). 110 23

Restricted chymotrypsin digestion of calf thymus H1 histone gives two fragments, residues 1--106 and 107--C-terminal. These were studied by proton magnetic resonance and circular dichroism. The N-terminal fragment exhibited some salt-induced structure in aqueous solution, but this did not parallel the globular structure of the intact H1 molecule. Comparison of circular dichroism results with helix predictions for this portion of the molecule suggests that the secondary structure may be the same in this fragment as it is in the corresponding region of the whole molecule. The C-terminal fragments show very little salt-induced structure. The N-terminal fragments binds to DNA very weakly, but the C-terminal fragment binds as strongly as the whole molecule. In the C-terminal fragment, about one quarter of the lysine residues are not bound to the DNA in water, but initial increase of salt concentration causes them to become bound. This increasing binding occurs under the same ionic conditions that cause chromatin condensation and condensation of H1 - DNA complexes, and it is suggested that there may be a connection between these phenomena.
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PMID:Studies on the role and mode of operation of the very-lysine-rich histone H1 (F1) in eukaryote chromatin. The properties of the N-terminal and C-terminal halves of histone H1. 117 57

The structure of octylcarbamoyl-alpha-chymotrypsin to a resolution of 3.0 A is described. The n-octyl side chain of the active site directed irreversible inactivator octyl isocyanate is bound exclusively in the hydrophobic substrate binding pocket. The n-octyl isocyanate forms a planar urethane bond with the Ser-195 Ogamma and extends approximately 1 A deeper into the hydrophobic pocket than the indolyl group of indoleacryloyl-alpha-chymotrypsin (Henderson, R. (1970), J. Mol. Biol. 54, 341). All the structural changes are essentially identical with those observed in indoleacryloyl-alpha-chymotrypsin including the observation of a hydrogen bonded water molecule between the carbonyl oxygen of the octylcarbamoyl group and the imidazole group of His-57. The observed mode of n-octyl alkyl binding to chymotrypsin is consistent with the hypothesis proposed earlier (Brown, W. E. and Wold, F. (1973), Biochemistry 12, 828).
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PMID:Alkyl isocyanates as active site-specific reagents for serine proteases. Location of alkyl binding site in chymotrypsin by X-ray diffraction. 119 30

The surface of the alpha-chymotrypsin globule is investigated using a three-dimensional model of the molecule, constructed on the basis of X-ray data by sectioning the space of the protein globule in cubic elements with a step of 3 A. The surface layer contains about 55% of the overall globule volume. The atomic density of so defined surface was found to be approximately equal to that in the inner part of the globule. Topographical maps of the alpha-chymotrypsin surface were drawn and an analysis of the distribution of polar and unpolar atoms and groups on the surface and in the inner part of the globule was carried out. Some conclusions drawn from the atomic density, energetic and structural heterogeneity of the surface and concerning the conformational integrity and functional activity of alpha-chymotrypsin molecule are presented. Some aspects of the protein hydration problem are discussed and a structural model of the alpha-chymotrypsin hydratation shell is proposed, the main features of which are amorphism and the lack of long-range effect on the structure of water around the hydrated protein globule.
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PMID:[The surface layer of the protein globule. Hydration of the alpha-chymotrypsin molecule]. 121 14

A variety of azobenzene compounds having bis-quaternary nitrogens have been shown to accelerate the hydrolysis by chymotrypsin of certain specific substrates by an allosteric mechanism. One of the most potent, 2,2'-bis[alpha-(benzyldimethylammonium)methyl]azobenzene dibromide (2,2'-QBzl) accelerated the hydrolysis of glutaryl-L-phenylalanine p-nitroanilide 40-fold at saturating concentration. Acceleration was by increasing kcat without altering Km. The hydrolysis of acetyl-L-tyrosine p-nitroanilide and acetyl-L-tyrosine anilide was also accelerated by Q-Bzl (25-fold and 1.8-fold respectively) while the hydrolysis of hemoglobin, azocoll and a number of esters was not affected. The inactivation of chymotrypsin by diphenylcarbamyl chloride and diphenylcarbamyl fluoride was accelerated by 2,2'-Q-Bzl. Reac;ivation in the presence of NH2OH was also accelerated, but in the absence of added nucleophile (i.e. of NH20H) no increase in rate was detectable. An allosteric effector was covalently attached to chymotrypsinogen A by reaction with 2,2'-bis[alpha-(o-bromomethylbenzyldimethylammonium)methyl]azobenezene dibromide. The product, when converted to active enzyme, was about 4 times more active than chymotrypsin as a result of an increase in kcat of hydrolysis; Km was unaffected. The mechanism of the allosteric acceleration process is not known but, because for all of the substrates affected acylation of the enzyme is rate-limitimg, it is tentatively suggested that the effectors facilitate proton transfer to the leaving group by an inductive effect on the 'charge relay system'. Spectral studies indicate that the allosteric site is a portion of the enzyme with a polarity near that of water, possibly on the outside surface of the enzyme molecule.
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PMID:Allosteric activation of the hydrolysis of specific substrates by chymotrypsin. 124 86

The synthetic cyclic tetrapeptide (L-Leu-L-Tyr-delta-Avaler-delta-Avaler) is an effective inhibitor of chymotrypsin, competitive with linear peptides like Ac-L-Leu-L-Tyr-OMe. An x-ray diffraction analysis of the crystal structure of the cyclic peptide shows that the conformation of the 18-membered ring is very similar to that of one of the four conformers of cyclic hexaglycyl. There is no internal hydrogen bonding. Side chains are located on two "corners" of the approximately rectangular ring. The chii1 angles for Leu and Tyr are -74 and -48 degrees, respectively. The Leu side chain is extended away from the polypeptide ring while the Tyr side chain is folded under an adjacent carbonyl bond. The cell parameters for the space group P2U are: a = 9.361 (3 A, b = 19.039 (10) A, c = 9.603 (3), A, and beta = 116.54 (3) degrees. A molecule of (CH3)2SO (disordered) and a molecule of H2O cocrystallized with the cyclic peptide.
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PMID:Conformation of cyclo-(L-Leu-L-Tyr-delta-Avaler-delta-Avaler), a synthetic inhibitor of chymotrypsin, by x-ray analysis. 124 91

Band 3 is the major, membrane-spanning, approximately90 000 dalton polypeptide of the human erythrocyte membrane. To facilitate the analysis of its structural integration into the membrane, we have cleaved this protein in situ into large fragments and ascertained their disposition. Digestion of intact cells with chymotrypsin yielded band 3 fragments with apparent molecular weights of 38 000 and 55 000. Both fragments resisted elution by NaOH and acetic acid, suggesting that they are anchored in the apolar core of the membrane. Both pieces communicate with the extracellular space, and the 55 000 dalton species extends to the cytoplasmic surface as well. Digestion of unsealed ghosts with chymotrypsin produced a hydrophobic 17 000 dalton species, a segment of the 55 000 dalton fragment, which spans and is firmly anchored in the core of the membrane. Trypsin and papain at low concentration generated integral band 3 fragments of 52 000 daltons and released major band 3 fragments of less than or equal to 41 000 daltons from the cytoplasmic side of the membrane. The latter water-soluble polypeptides remained associated in discrete complexes which retained the capacity to bind glyceraldehyde-3-phosphate dehydrogenase. An interchain disulfide bond, which can be induced only at the cytoplasmic surface, cross-linked intact band 3, and certain of its water-soluble fragments. Finally, fragments of 23 000 daltons were generated from the innersurface domain by reacting disulfide-linked band 3 dimers with cyanide or reduced polypeptides with 2-nitro-5-thiocyanobenzoate. A provisional ordering of these fragments is proposed.
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PMID:Proteolytic dissection of band 3, the predominant transmembrane polypeptide of the human erythrocyte membrane. 125 33


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