Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.4.24.27 (thermolysin)
 1,894 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

1. The reactivities of phenylglyoxal (PGO), glyoxal (GO), and/or methylglyoxal (MGO) with several proteins, including ribonuclease A [EC 3.1.4.22] and its derivatives, alpha-chymotrypsin [EC 3.4.21.1], trypsin [EC 3.4.21.4], lysozyme [EC 3.2.1.17], pepsin [EC 3.4.23.1], rennin [EC 3.4.23.4], thermolysin, and insulin and its B chain, have been examined. From analyses of the reaction products, PGO was shown to be the most specific for arginine residues. GO and MGO also reacted rapidly with arginine residues, but they also reacted with lysine residues to a significant extent. A side reaction with N-terminal alpha-amino groups was observed with each of these reagents. 2. Two arginine residues out of four in ribonuclease A, two out of three in alpha-chymotrypsin, one out of two in trypsin, one out of two in pepsin, and one out of five in rennin appeared to react with PGO fairly rapidly, indicating a difference in the relative accessibility of these residues by the reagent. Extensive modification of the arginine residues by PGO occurred with RCM-derivatives of ribonuclease A and insulin B chain. The N-terminal isoleucine residues of alpha-chymotrypsin and trypsin appeared to be unreactive with PGO because of salt bridge formation with an aspartyl residue. The activity of alpha-chymotrypsin toward N-benzoyl-L-tyrosine ethyl ester and the lytic activity of lysozyme were lost rapidly on treatment with PGO, as in the case of ribonuclease A. Pepsin and rennin were only partially inactivated by reaction with PGO.
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PMID:Further studies on the reactions of phenylglyoxal and related reagents with proteins. 32 41

A method was devised to isolate N-terminal peptide fragments from the polypeptide chains constituting thyroglobulin even in the case when the terminal amino groups are naturally blocked, for instance, acylated. Reduced and carboxymethylated hog thyroglobulin was first acetylated and digested with thermolysin. The blocked N-terminal peptide fragments were separated from the unblocked N-terminal fragments by column chromatography on Dowex 50, then on Dowex 1 after dinitrophenylation, and finally fractionated into ten fractions by paper chromatography after gel filtration on Sephadex G-10. Structural analyses by enzymic or partial acid hydrolysis of these peptide fractions failed to detect N-terminal acetyl amino acid. Instead, pyroglutamyl peptides including pyroglutamylleucine were found. By the same method, acetylated lysine and glycine were identified for chicken lysozyme and horse myoglobin, respectively. The use of thermolysin because of its unique specificity, and the possible relevance of the present result to the previous data on the N-terminal analysis of thyroglobulin are discussed.
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PMID:The presence of N-terminal pyroglutamyl residues in hog thyroglobulin. 93 62

Circular dichroism spectra have been obtained for albumin, alpha-chymotrypsinogen, collagen, concanavalin A, elastase, hemoglobin, histone f2b, alpha-lactalbumin, lactate dehydrogenase, beta-lactoglobulin, lysozyme, myoglobin, papain, ribonuclease A, and thermolysin in the presence of sodium dodecyl sulfate and dithiothreitol. While all spectra have the shape anticipated for a mixture of random coil and alpha helix, the intensities differ markedly ([theta]222 ranges from --1400 to --15 000 deg cm2/dmol). The variation in the circular dichroism can be quantitatively explained by a model which assumes that the arginyl, histidyl, and lysyl residues have an enhanced probability of propagating a helical segment in the presence of the detergent. The model also permits the computation of dimensional properties (unperturbed end-to-end distance and radius of gyration) for polypeptides of known amino acid sequence. Such computations have been performed for 67 proteins. The computed dimensions are compatible with experimental values and with the molecular weight dependence of the transport properties of the complexes. Furthermore, the model can account for the abnormal transport properties of the sodium dodecyl sulfate complexes formed by ribonuclease A, collagen fragments, and histones f2a1, f2a2, f2b, and f3. Even though some of the protein--sodium dodecyl sulfate complexes have helical contents as high as 50%, their overall conformation more closely approximates that of a random coil than a rod.
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PMID:Conformational properties of the complexes formed by proteins and sodium dodecyl sulfate. 96 36

The flexibility plot of a protein lies on the observation that amino acid residues with the highest turn potential, i.e. located in highly mobile regions of protein surface, also possess the smallest volumes as well as the lowest hydrophobicities. The plot is generated by shifting a five residue window along the protein sequence and calculating the value of the hydrophobicity-volume product for consecutive quintuplets of amino acid residues. The concomitant occurrence of small volumes and low hydrophobicities results in very deep minima. A threshold value has also been introduced in order to discriminate significant minima. To substantiate the interpretation that the selected minima actually indicate very flexible segments of a protein (loops, turns, etc.), we have compared plots obtained for model proteins (lysozyme, myoglobin, ribonuclease, trypsin, thermolysin and T4 lysozyme) with X-ray thermal factors profiles available for the same proteins. When compared to thermal profiles, the majority of flexible segments evidenced by our plots have been found to be in agreement with regions characterized by high thermal factors. Results have also been discussed in the light of local organization possessed by examined proteins.
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PMID:Flexibility plot of proteins. 274 66

A convenient method for the determination of unfolding rates of small globular proteins under physiological conditions was developed using digestion with proteases. The apparent first-order rate constants for digestion of lysozyme with thermolysin and with Pronase at pH 8 and 50 degrees C were shown to be saturated with increases of concentrations of these proteases. The maximum rate constants extrapolated were identical in digestions with two different proteases, and were found to be equal to the unfolding rate constant of lysozyme. Similarly, the unfolding rate constant of RNase A at pH 8 and 50 degrees C, and those of lysozyme, RNase A and beta-lactoglobulin at pH 8 and 40 degrees C, were determined by the digestion method. Thus, it was shown that digestion by proteases proceeds mainly via the unfolded state of proteins.
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PMID:Unfolding rates of globular proteins determined by kinetics of proteolysis. 378 15

The disulfide peptides from the tryptic digestion of cyanogen bromide-treated hen egg white lysozyme (HEWL) were isolated by reverse phase high performance liquid chromatography (HPLC) and identified by amino acid analysis. Three peptides containing the I-VIII, II-VII, and III-V + IV-VI disulfide bonds were obtained. The two-disulfide peptide was further digested with proline-specific endopeptidase (PCE) (EC 3.4.21.26). Amino acid analysis of digest peptides separated by HPLC showed four peptides with the IV-VI disulfide bond as well as a peptide with the III-V disulfide bond. The IV-VI peptides were produced by hydrolysis of several alanine-X bonds as well as the prolyl-cystine bond. Our studies show that alanyl peptide bonds to lysyl, seryl, and leucyl residues are susceptible to hydrolysis by PCE preparations, thus substantially extending its known specificity range. The two-disulfide peptide was also digested sequentially with thermolysin and PCE; the resulting IV-VI and III-V peptides were identified by HPLC and amino acid analysis. PCE showed substantial activity at pH 5.3 as well as at pH 8.3. The lower pH is useful in studies of proteins or peptides where base-catalyzed reactions must be limited.
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PMID:Use of proline-specific endopeptidase in the isolation of all four "native" disulfides of hen egg white lysozyme. 390 90

Proteolysis of hen egg-white lysozyme by thermolysin in 50% aqueous trifluoroethanol for 6-24 h at 40-52 degrees C produces a 'nicked' protein species which was purified to homogeneity by reverse-phase HPLC and characterized. Protein chemistry analytical methods were used to establish that thermolysin cleaves the 129-residue chain of lysozyme at peptide bond Lys97-Ile98. Nicked lysozyme, which is therefore constituted by fragments 1-97 and 98-129 cross-linked by disulfide bonds, was approximately 20% and 60% active towards Micrococcus luteus cells in respect to native intact lysozyme when assayed at 25 degrees C or 5 degrees C, respectively. Circular dichroic measurements provided evidence that nicked lysozyme in aqueous buffer at low temperature maintains the secondary structure content of native lysozyme, whereas the microenvironment of the aromatic chromophores, in particular of tryptophan residue(s), was somewhat perturbed. The stability to heat and urea denaturation of nicked lysozyme was dramatically reduced with respect to that of the intact protein. For example, the tm of the nicked species was 28 degrees C in comparison with 73 degrees C for the unmodified enzyme, both at pH 7.0. Inspection of the X-ray structure of hen lysozyme reveals that thermolysin cleaves at the C-terminus of alpha-helix C (residues 88-98) located at the interface of the two structural domains of the protein, thus destabilizing the helix dipole and disrupting important tertiary interactions of the native enzyme. These results were interpreted considering that lysozyme in 50% aqueous trifluoroethanol is an expanded and flexible protein species largely maintaining native-like secondary structure, but lacking tertiary interactions [Buck, M., Radford, S. E. & Dobson, C. M. (1993) Biochemistry 32, 669-678]. Thus, whereas native lysozyme in its well-packed and rigid structure is quite resistant to proteolysis and only upon thermal unfolding is degraded to many small peptides in an all-or-none process, lysozyme in the trifluoroethanol state is sufficiently flexible to act as a substrate for the protease, but maintains significant secondary structure (helix) precluding extensive proteolytic degradation.
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PMID:Limited proteolysis of lysozyme in trifluoroethanol. Isolation and characterization of a partially active enzyme derivative. 760 52

The lysozyme of bacteriophage T7 is a bifunctional protein that cuts amide bonds in the bacterial cell wall and binds to and inhibits transcription by T7 RNA polymerase. The structure of a mutant T7 lysozyme has been determined by x-ray crystallography and refined at 2.2-A resolution. The protein folds into an alpha/beta-sheet structure that has a prominent cleft. A zinc atom is located in the cleft, bound directly to three amino acids and, through a water molecule, to a fourth. Zinc is required for amidase activity but not for inhibition of T7 RNA polymerase. Alignment of the zinc ligands of T7 lysozyme with those of carboxypeptidase A and thermolysin suggests structural similarity among the catalytic sites for the amidase and these zinc proteases. Mutational analysis identified presumed catalytic residues for amidase activity within the cleft and a surface that appears to be the site of binding to T7 RNA polymerase. Binding of T7 RNA polymerase inhibits amidase activity.
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PMID:The structure of bacteriophage T7 lysozyme, a zinc amidase and an inhibitor of T7 RNA polymerase. 817 Oct 31

A backbone-dependent rotamer library for amino acid side-chains is developed and used for constructing protein side-chain conformations from the main-chain co-ordinates. The rotamer library is obtained from 132 protein chains in the Brookhaven Protein Database. A grid of 20 degrees by 20 degrees blocks for the main-chain angles phi, psi is used in the rotamer library. Significant correlations are found between side-chain dihedral angle probabilities and backbone phi, psi values. These probabilities are used to place the side-chains on the known backbone in test applications for six proteins for which high-resolution crystal structures are available. A minimization scheme is used to reorient side-chains that conflict with the backbone or other side-chains after the initial placement. The initial placement yields 59% of both chi 1 and chi 2 values in the correct position (to within 40 degrees) for thermolysin to 81% for crambin. After refinement the values range from 61% (lysozyme) to 89% (crambin). It is evident from the results that a single protein does not adequately test a prediction scheme. The computation time required by the method scales linearly with the number of side-chains. An initial prediction from the library takes only a few seconds of computer time, while the iterative refinement takes on the order of hours. The method is automated and can easily be applied to aid experimental side-chain determinations and homology modeling. The high degree of correlation between backbone and side-chain conformations may introduce a simplification in the protein folding process by reducing the available conformational space.
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PMID:Backbone-dependent rotamer library for proteins. Application to side-chain prediction. 846 64

High hydrostatic pressures in the biologically relevant range (< or = 1,200 bar) are known to cause dissociation of oligomeric enzymes in vitro, whereas protein denaturation requires pressures far beyond this range. Pressure-induced inactivation phenomena attributable to neither of these effects are shown to occur in monomeric enzymes. Three different types of pressure dependence can be distinguished: (1) a linear dependence of catalytic rate constants on pressure, as predicted by the activated complex theory, observed for lysozyme and thermolysin; (2) a biphasic profile consisting of two linear contributions, found for trypsin; (3) maximum curves, as observed for both directions of the octopine dehydrogenase reaction. The third case may be ascribed to a pressure-induced decrease in the partial specific volume of the protein, resulting in reduced flexibility of the active site. This mechanism may also apply to the pressure-induced inactivation of assembly systems stabilized against dissociation in the cell.
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PMID:The catalytic activities of monomeric enzymes show complex pressure dependence. 847 59


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