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Query: UNIPROT:P06889 (Mol)
 630,302 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Reversible unfolding of bovine chymotrypsinogen A in 2H2O either by heating at low pH or by exposure to 6 M guanidinium chloride results in the exchange of virtually all the nitrogen-bound hydrogens that give rise to low-field 1H NMR peaks, without significant exchange of the histidyl ring Cepsilon1 hydrogens. These preexchange procedures have enabled the resolution of two peaks, using 250-MHz correlation 1H NMR spectroscopy, that are attributed to the two histidyl residues of chymotrypsinogen A. Assignments of the Cepsilon1 hydrogen peaks to histidine-40 and -57 were based on comparison of the NMR titration curves of the native zymogen with those of the diisopropylphosphoryl derivative. Two histidyl Cepsilon1 H peaks were also resolved with solutions of preexchanged chymotrypsin Aalpha. The histidyl peaks of chymotrypsin Aalpha were assigned by comparison of NMR titration curves of the free enzyme with those of its complex with bovine pancreatic trypsin inhibitor (Kunitz). The NMR titration curves of histidine-57 in the zymogen and enzyme and histidine-40 in the zymogen exhibit two inflections; the additional inflections were assigned to interactions with neighboring carboxyl groups: aspartate-102 in the case of histidine-57 and aspartate-194 in the case of histidine-40 of the zymogen. In bovine chymotrypsinogen A in 2H2O at 31 degrees C, histidine-57 has a pK' of 7.3 and aspartate-102 a pK' of 1.4, and the histidine-40-aspartate-194 system exhibits inflections at pH 4.6 and 2.3. In bovine chymotrypsin Aalpha under the same conditions, the histidine-57-aspartate-102 system has pK' values of 6.1 and 2.8, and histidine-40 has a pK' of 7.2. The results suggest that the pK' of histidine-57 is higher than the pK' of aspartate-102 in both zymogen and enzyme. A significant difference exists in the structure and properties of the catalytic center between the zymogen and activated enzyme. In addition to the difference in pK' values, the chemical shift of histidine-57, which is highly abnormal in the zymogen (deshielded by 0.6 ppm), becomes normalized upon activation. These changes may explain part of the increase in the catalytic activity upon activation. The 1H NMR chemical shift of the Cepsilon1 H of histidine-57 in the chymotrypsin Aalpha-pancreatic trypsin inhibitor (Kunitz) complex is constant between pH 3 and 9 at a value similar to that of histidine-57 in the porcine trypsin-pancreatic trypsin inhibitor complex [Markley, J.L., and Porubcan, M. A. (1976), J. Mol. Biol. 102, 487--509], suggesting that the mechanisms of interaction are similar in the two complexes.
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PMID:Zymogen activation in serine proteinases. Proton magnetic resonance pH titration studies of the two histidines of bovine chymotrypsinogen A and chymotrypsin Aalpha. 3 98

The alterations of tryptophan fluorescence parametres with pH may be due to: 1) conformational changes; 2) changes in the ionic state of groups capable of quenching the tryptophan fluorescence. The applications of the model of discrete forms of tryptophan allow one to separate these mechanisms and estimate the middle points of conformational changes and pK's of quenching groups. For chymotrypsin (CT) and chymotrypsinogen (CTG) conformational changes were registrated with middle points: CT pH 4.1 and 8.8; CTG -- pH 3.2 and 9.8, and pK's of histidines: CT -- 5.4 and 6.6; CTG -- 5.6 and 7.0. For trypsin conformational changes were shown with middle points: pH 3.2; 5.8; 8.5 and for lysozyme -- pH 5.9.
Mol Biol (Mosk)
PMID:[pH-dependence of fluorescence parameters of chymotrypsin, chymotrypsinogen, trypsin and lysozyme]. 3 49

Flounder muscle (Pseudopleuronectes americanus) glyceraldehyde-3-phosphate dehydrogenase was characterized as to its stability towards various inactivating treatments in the presence and absence of the enzyme cofactor, NAD. Incubation of a partially purified enzyme preparation at urea concentrations greater than 2 M produced a very rapid inactivation. NAD greatly reduced the rate of inactivation at all the urea concentrations tested. Incubation of each of the three major muscle enzyme forms in 0.1 percent trypsin or chymotrypsin for forty-five minutes decreased the activity of each form by 65 percent and 55 percent, respectively. NAD (5mM) afforded complete protection to each enzyme form from proteolytic digestion by these two enzymes. Exposure of each form to 50 degrees or 20 mM ATP also led to gross inactivation which could be greatly reduced if the respective incubations were performed in the presence of 5mM NAD. NAD was also found to be required for the renaturation of the unfolded urea-denatured subunits to form the active tetramer.
Mol Cell Biochem 1975 Sep 30
PMID:Effect of NAD on flounder muscle glyceraldehyde 3-phosphate dehydrogenase. 17 55

To collect information on synthesis and regulation of the peptidoglycan-associated pore-forming outer membrane proteins b and c, mutants resistant to phages Me1 and TuIa were analyzed. Genetic analysis showed three linkage groups, corresponding with the genes tolF (phenotype b-c+), meoA (phenotype b+c-) and ompB (phenotypes b-c-, b-c+, b++c- and b++c+/-). It has recently been described that also a b+c- phenotype can occur in the latter linkage group [Chai, T., Foulds, J., J. Bacteriol. 130, 781-786 (1977)]. Among ompB (b-c+)/meoA (b+c-) double mutants strains were found with the b+c- phenotype, showing that ompB is not the structural gene for protein b. Studies on purified proteins b and c showed profound differences between the two proteins with respect to the electrophoretic mobility of fragments obtained by treatment with cyanogen bromide, trypsin and chymotrypsin. The amino acid in position three of the amino-termini of proteins b and c, isolated from isogenic strains, were identified as isoleucine and valine respectively. Both the genetic and biochemical results are consistent with a model recently published [Ichihara, S., Mizushima, S., J. Biochem. (Japan) 83, 1095-1100 (1978)] which predicts that tolF and meoA are the structural genes for the proteins b and c respectively and that ompB is a regulatory gene whose product regulates the levels of both proteins.
Mol Gen Genet 1979 Jan 31
PMID:Genetics and biochemistry of the peptidoglycan-associated proteins b and c of Escherichia coli K12. 37 3

The isolation and characterization of two mutants of Escherichia coli K12 with an altered outer membrane protein c is described. The first mutant, strain CE1151, was isolated as a bacteriophage Me1 resistant strain which contains normal levels of protein c. Mutant cells adsorbed the phage with a strongly decreased rate. Complexes of purified nonheat modified wild type protein c and wild type lipopolysaccharide inactivated phage Me1, indicating that these components are required for receptor activity for phage Me1. When wild type protein c was replaced by protein c of strain CE1151, the receptor-complex was far less active, showing that protein c of strain CE1151 is altered. The second mutant produces a protein c with a decreased electrophoretic mobility, designated as protein c. An altered apparent molecular weight was also observed for one or more fragments obtained after fragmentation of the mutant protein with cyanogen bromide, trypsin and chymotrypsin. Alteration of protein c was not accompanied by a detectable alteration in protein b or its fragments. Both mutations are located at minute 48 of the Escherichia coli K12 linkage map. The results strongly suggest that meoA is the structural gene for protein c.
Mol Gen Genet 1979 Jan 31
PMID:meoA is the structural gene for outer membrane protein c of Escherichia coli K12. 37 4

After polyadenylation in vitro of the influenza virus RNA segment which contains the coding information for the matrix protein, a cDNA copy can be made using the primer p(dT)8-dA and reverse transcriptase. The sequence of 166 nucleotides of the cDNA was determined by a modification [Brownlee, G. G. & Cartwright, E. M. (1977) J. Mol. Biol, 114, 93--117] of the plus/minus method [Sanger, F. & Coulson, A. R. (1975) J. Mol. Biol. 94, 441--481] and adaptation of the "dideoxy" method [Sanger, F., Nicklen, S. & Coulson, A. R. (1977) Proc. Natl. Acad. Sci. U.S.A. 74, 5463--5467] for sequencing DNA. The cDNA sequences is of the same sense as the mRNA for matrix protein and contains a potential initiating codon, d(ATG), at position 26--28. When matrix protein purified from virus particles was digested with chymotrypsin or trypsin and the amino acid compositions of separated peptides determined, one peptide containing nine amino acids found which had a composition corresponding to that predicted by the cDNA sequence following the first methionine codon, confirming that protein synthesis initiates at this position. The compositions of four other peptides matches those predicted from the nucleotide sequence. There is no processing of the N terminus of the protein before incorporation into the virus particle except for removal of the N-terminal methionine and addition of a "blocking" group on the resulting N-terminal serine residue.
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PMID:Nucleotide sequence coding for the N-terminal region of the matrix protein influenza virus. 57 97

The heavy water (D2O) has been shown to induce the conformational transitions in trypsin, chymotrypsin and pepsin. The transfer of proteins from H2O into D2O results a change in their sensitivity to UV-light. An increase in sensitivity to the irradiation at 248 nm and a decrease in sensitivity to the irradiation at 280 nm were observed. The quantum yield of chromophore photolysis (for cystyne and tryptophan) is correspondingly changed. However, although the quantum yield of sensitized reduction of cystine by solvated electrons photochemically ejected from the aromatic acid residues during irradiation at 280 nm increases instead of a rise a drop in the quantum yield of protein inactivation is registered. The data obtained are discussed in terms of importance of solvated shell for conformational stability of proteins. The solvated electrons are suggested to be transfered mainly to nonessential disulfide bridges within trypsin molecule. Rupture of these bonds does not result in trypsin inactivation.
Mol Biol (Mosk)
PMID:[Influence of heavy water (D20) on the conformation and UV-sensitivity of proteins]. 80 85

We find that specific oxidation for the Met-192 residue in delta-chymotrypsin to methionine sulfoxide results in a twofold increase in Km(app) and unchanged kcat in the hydrolysis of N-acetyl mono(amino acid) amide substrates. However, the catalyzed hydrolyses of N-acetyl dipeptide amide substrates by (methionine sulfoxide)-192-delta-chymotrypsin (MS-delta-Cht) shows a four- to fivefold decrease in kcat and unchanged Km(app) with respect to delta-chymotrypsin. Hydrolysis of alpha-casein by MS-delta-Cht shows a similar 4.2-fold decrease in kcat. These results imply that the Met-192 acts differently with substrates that bind only in the primary, S1, binding site (i.e., AcPheNH2) from those that bind to more extended regions of the enzyme active site. In the binding of c+AcPheNH2 and AcTrpNH2, the results support a mechanism in which the Met-192 acts to slow the rate of sustrate dissociation from the Michaelis complex to free substrate and enzyme. This is in agreement with the x-ray crystallographic structure of dioxane inhibited alpha-chymotrypsin (Steitz, T., et al. (1969), J. Mol. Biol. 46, 337). However, this mechanism is not apparent when peptide and protein substrates bind. The decrease in kcat on Met-192 modification of approximately fivefold in the hydrolysis of polypeptide substrates show a small, but significant, catalytic contribution of the Met-192 toward the lowering of the energy of activation polypeptide substrate hydrolysis by chymotrypsin. This may support the crystallographic model of Fersht et al. (Fersht, A., et al. (1973), Biochemistry 12, 2035) in which it is proposed that the Met-192 participates in the distortion of bound polypeptide substrates toward the reaction transition-state configuration and, thus, plays a role in catalysis. However, if this mechanism occurs, the effect is small, only contributing about 1 kcal/mol to the lowering of the reaction activation energy.
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PMID:The role of methionine-192 of the chymotrypsin active site in the binding and catalysis of mono(amino acid) and peptide substrates. 96 30

Three to five isozymes of pancreatic proteinase exist in mice, and they have been designated as bands I, II, III, IV, and V. Identification experiments of these isozymes were carried out in this study; bands I, IV, and V are trypsin, and bands II and III are chymotrypsin. Therefore, it is concluded that Prt-1, controlling band V, is a locus for trypsin and Prt-2, controllong bands II and III, is a locus for chymotrypsin. In addition, a new locus, Prt-3, has been found. At this locus the two allelic genes, Prt-3a and Prt-3b, control the low and high tryptic activities of band IV, respectively. Prt-3 is present only in the strain Mol-A. Linkage experimentation has shown that Prt-1 is closely linked to Prt-3.
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PMID:Genetic study of pancreatic proteinase in mice (Mus musculus): genetic variants of trypsin and chymotrypsin. 100 1

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


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