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Query: EC:3.2.1.17 (lysozyme)
 21,489 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The complex formed between hen egg white lysozyme (HEL) and the monoclonal antibody HyHEL-10 Fab fragment has an interface composed of van der Waals interactions, hydrogen bonds, and a single ion pair. The antibody overlaps part of the active site cleft. Putative critical residues within the epitope region of HEL, identified from the x-ray crystallographic structure of the complex, were replaced by site-directed mutagenesis to probe their relative importance in determining affinity of the antibody for HEL. Twenty single mutations of HEL at three contact residues (Arg-21HEL, Asp-101HEL, and Gly-102HEL) and at a partially buried residue (Asn-19HEL) in the epitope were made, and the effects on the free energies of dissociation were measured. A correlation between increased amino acid side-chain volume and reduced affinity for HELs with mutations at position 101 was observed. The D101GHEL mutant is bound to HyHEL-10 as tightly as wild-type enzyme, but the delta delta Gdissoc is increased by about 2.2 kcal (9.2 kJ)/mol for the larger residues in this position. HEL variants with lysine or histidine replacements for arginine at position 21 are bound 1.4-2.7 times more tightly than those with neutral or negatively charged amino acids in this position. These exhibit 1/40 the affinity for HyHEL-10 Fab compared with wild type. There is no side-chain volume correlation with delta delta Gdissoc at position 21. Although Gly-102HEL and Asn-19HEL are in the epitope, replacements at these positions have no effect on the affinity of HEL for the antibody.
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PMID:High-resolution mapping of the HyHEL-10 epitope of chicken lysozyme by site-directed mutagenesis. 768 15

The specificity of hen egg-white lysozyme (HEL)-reactive rabbit antibodies induced by the peptide loop I.II (sequences 57-107 containing Cys64-Cys80 and Cys76-Cys94) of HEL was clarified by analyzing their cross-reactions with various avian lysozymes and their reaction with synthetic peptides (sequences 59-82) in which alanine was substituted for the amino acid at certain positions. The Arg-68 residue of HEL plays a dominant role in the binding, while Gly-71, Ser-72, Arg-73, and Pro-79 also contribute to the binding of two anti-Ploop I.II antibodies (rabbit number 125 and 126). These residues, although remote in sequence, are grouped together in the crystal structure of HEL and may form an area of contact with the antibody. Contributions by Trp-63, Ile-78, and Asn-77 to the binding of the two antibodies to HEL were excluded. These results support the idea that the anti-Ploop I.II antibodies recognize a conformational type of epitope which is similar to that of native HEL. The immunogenicity of the reduced and alkylated form of Ploop I.II was also tested, but it failed to induce an HEL-reactive antibody.
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PMID:Characteristics of the epitope of protein-reactive anti-peptide antibodies. 768 42

Protein disulfide isomerase (PDI), which catalyses the folding of newly synthesized or denatured proteins through correct disulfide formation, was purified from soybean (Glycine max). The enzyme was purified 12,000-fold over crude extracts to apparent homogeneity in six purification steps: 60-70% ammonium sulfate fractionation, and chromatography on DEAE Toyopearl 650M, Q-Sepharose Fast Flow, Hiload Superdex 200 pg, Phenyl Sepharose HP, and TSK G-3000 SW. The native enzyme had a molecular weight of 120 kDa on gel filtration. Subunit molecular weight was estimated as 63 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis, thus indicating the enzyme to be comprised of two identical subunits. The enzyme pH optimum was 8.0 with reactivation of scrambled RNase, and the pI 7.65. The N-terminal amino acid sequence of soybean PDI was homologous to that of mature alfalfa as deduced from the cDNA sequence. Two identical active site sequences, APWCGHCK, were obtained from different proteolytic peptide fragments of soybean PDI. Soybean PDI facilitated reactivation not only of scrambled RNase, but denatured and reduced lysozyme and the Bowman Birk soybean trypsin inhibitor as well. This is the first report to appear on the the purification, characterization and amino acid sequence analysis of the active site of a plant PDI.
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PMID:Purification and characterization of protein disulfide isomerase from soybean. 777 91

A gamma-glutamyl peptide-hydrolysing enzyme was partially purified from Actinobacillus actinomycetemcomitans. The enzyme required metal ions, and 1 to 2 mM Mn2+ ions, especially, were essential for hydrolytic reaction. Its distribution by treatment of cells with lysozyme-EDTA suggested that the enzyme was a membrane-bound protein. The pI of the enzyme was in range of pH 5.1 to 5.6, and the apparent Km value for gamma-glutamyl-p-nitroanilide was 3.0 x 10(-4) M. The enzyme hydrolysed specifically gamma-glutamyl residues from the N-terminal of gamma-glutamyl compounds such as gamma-Glu-Met, gamma-Glu-Ala, gamma-Glu-Leu and gamma-Glu-Tyr, but did not catalyse the transpeptidation reaction. Neither free amino acids such as Ala-, Pro-, Gly- and Leu-p-nitroanilide nor alpha-glutamyl derivatives were hydrolysed. Its activity was strongly inactivated by metal chelators (EDTA or o-phenanthroline) and amino acids (Glu, Gln). In addition, the activity was specifically inactivated by gamma-glutamyl affinity-labelling reagents such as AT-125, 6-diazo-5-oxo-L-norleucine and azaserine, which are inhibitors of the gamma-glutamyl donor sites of mammalian gamma-glutamyl transpeptidase. Antibodies against bovine kidney gamma-glutamyl transpeptidase decreased the activity of the bacterial enzyme by 65%. These results suggested that the active sites in the bacterial enzyme were similar to those in mammalian gamma-glutamyl transpeptidase.
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PMID:Partial purification and some properties of gamma-glutamyl peptide-hydrolysing enzyme from Actinobacillus actinomycetemcomitans. 779 32

The stability changes caused by single amino acid substitutions are studied by a simple, empirical method which takes account of the free energy change in the compact denatured state as well as in the native state. The conformational free energy is estimated from effective inter-residue contact energies, as evaluated in our previous study. When this method is applied, with a simple assumption about the compactness of the denatured state, for single amino acid replacements at Glu49 of the tryptophan synthase alpha subunit and at Ile3 of bacteriophage T4 lysozyme, the estimates of the unfolding Gibbs free energy changes correlate well with observed values, especially for hydrophobic amino acids, and it also yields the same magnitudes of energy as the observed values for both proteins. When it is also applied for amino acid replacements at various positions to estimate the average number of contacts at each position in the denatured state from the observed value of unfolding free energy change, those values for replacements with Gly and Ala at the same residue position in staphylococcal nuclease correlate well with each other. The estimated numbers of contacts indicate that the protein is not fully expanded in the denatured state and also that the compact denatured state may have a substantially native-like topology, like the molten globule state, in that there is a weak correlation between the estimated average number of contacts at each residue position in the denatured state and the number of contacts in the native structure. These results provide some further evidence that the inter-residue contact energies as applied here (i) properly reflect actual inter-residue interactions and (ii) can be considered to be a pairwise hydrophobicity scale. Also, the results indicate that characterization of the denatured state is critical to understanding the folding process.
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PMID:Protein stability for single substitution mutants and the extent of local compactness in the denatured state. 785 36

To examine the effect of a conformational constraint introduced into the Arg-Gly-Asp (RGD) sequence on cell adhesion activity, we constructed a mutant protein by inserting an RGD-containing sequence flanked by two Cys residues between Val74 and Asn75 of human lysozyme. The CRGDSC-inserted lysozyme was expressed in yeast, purified, and designated as Cys-RGD4. Using baby hamster kidney cells, Cys-RGD4 was shown to possess even higher cell adhesion activity than that of the RGDS-inserted lysozyme, RGD4. The Cys-RGD4 protein was co-crystallized with a lysozyme inhibitor, tri-N-acetylchitotriose, and the three-dimensional structure was determined at 1.6-A resolution by x-ray crystallography. In contrast to RGD4, the inserted RGD-containing region of Cys-RGD4 was well defined. The structural analysis revealed that the two inserted Cys residues form a new disulfide bond in Cys-RGD4, as expected, and that the RGD region assumes a type II' beta-turn conformation of Gly-Asp with a hydrogen bond between the C = O of Arg and the H-N of Ser. In addition, it was confirmed that two more hydrogen bonds are present in the RGD region of the Cys-RGD4 lysozyme. These results suggest that the conformation of the RGD-containing region is rigid and stable in the Cys-RGD4 molecule and that the type II' beta-turn structure of RGD is essential for binding to integrins with high affinity.
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PMID:Structure of a conformationally constrained Arg-Gly-Asp sequence inserted into human lysozyme. 789 Jun 92

The mechanism of irreversible inactivation of lysozyme at pH 4, 100 degrees C, was investigated. It was elucidated that the inactivation was caused by production of molecules in an irreversibly denatured state. From analyses of the mechanism of production of the inactive enzyme, the inactivation was not evoked by a single chemical reaction. The free energy change between the folded and unfolded states decreased by the accumulation of chemical reactions (isomerization of Asp-Gly, deamidation of Asn, racemization of Asp and Asn, and cleavage of the Asp-X-peptide bond) induced at high temperature. Thus, certain molecules were ultimately in the unfolded state even at low temperature and lost activity. Moreover, a good correlation between the stability (free energy change) and the averaged number of chemical reactions that leads to the inactivation was obtained on the basis of some assumptions.
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PMID:The mechanism of irreversible inactivation of lysozyme at pH 4 and 100 degrees C. 794 8

For elucidating the contribution of structurally perturbed antigenic residues upon antibody binding to antigen-antibody interaction, the interaction between hen egg white lysozyme (HEL) and HyHEL10 Fv fragment, which is one of several monoclonal antibodies against HEL and structurally well defined (Padlan, E.A., Silverton, E. W., Sheriff, S., Cohen, G. H., Smith-Gill, S. J., and Davies, D. R. (1989) Proc. Natl. Acad. Sci. U. S. A. 86, 5938-5942), was investigated. Asp-101 and Trp-62 of HEL, whose conformations are perturbed by the binding of antibody HyHEL10 in this interaction, were replaced with Gly, and the resulting interactions were studied by assay of the inhibition of the lysozyme activity with the Fv fragment and by titration calorimetry. The results can be summarized as follows. 1) It was possible to prepare the fully functional Fv fragment of HyHEL10 using a secretory expression system in Escherichia coli. Its inhibition profile for HEL activity was almost indistinguishable from that of HyHEL10 IgG, and the contribution of enthalpy to driving the interaction was shown to be significant. 2) A thermodynamic study of the interaction between the D101G mutant HEL and the Fv fragment revealed that, although the negative enthalpy change was smaller than that for the wild type, the Gibbs energy was almost identical to that of the wild type, which resulted from the smaller entropy loss. 3) Study of the interaction between the W62G mutant HEL and this Fv fragment indicated that the rotation of the Trp-62 indole ring upon binding of the antibody made an enthalpic contribution to antibody-antigen interaction, although Trp-62 of HEL was proposed not to be the direct contact residue in the HyHEL10.HEL complex. 4) From these results, it was confirmed experimentally that structural perturbations of antigenic residues upon antibody binding of antigen would contribute to the gain of enthalpic energy, in spite of partial offset by entropic loss, and to driving the interaction.
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PMID:Contribution to antibody-antigen interaction of structurally perturbed antigenic residues upon antibody binding. 796 32

The solvation of polar groups at the N-terminal end of alpha-helices was studied by comparing the crystal structures of T4 lysozyme, barley chymotrypsin inhibitor 2 (CI2), barnase and their respective N-cap mutants. Whether or not the N3 residue is solvated on mutating the N-cap Thr/Ser to Ala or Gly appears to be related to the identities and the side-chain conformations of the N2 and N3 residues. When these two residues are alanines, as is in the pseudo-wild-type CI2 (E33A/E34A), the main-chain NH at the N3 position is exposed to the solvent and can be solvated. If the N2 residue is an Asp or a Glu, it is more likely that the side-chain of these residues will form a surrogate N-cap with the amide NH at N3 to compensate for the lost -OH group. In this case, no additional solvation will be observed. In general, Gly can be more stable than Ala at the N-cap because its small side-chain allows nearby polar groups to form hydrogen bonds with optimal geometry with solvent molecules or other polar groups.
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PMID:Stability and solvation of Thr/Ser to Ala and Gly mutations at the N-cap of alpha-helices. 803 23

The crystal structures of pheasant and guinea fowl lysozymes have been determined by X-ray diffraction methods. Guinea fowl lysozyme crystallizes in space group P6(1)22 with cell dimensions a = 89.2 A and c = 61.7 A. The structure was refined to a final crystallographic R-factor of 17.0% for 8,854 observed reflections in the resolution range 6-1.9 A. Crystals of pheasant lysozyme are tetragonal, space group P4(3)2(1)2, with a = 98.9 A, c = 69.3 A and 2 molecules in the asymmetric unit. The final R-factor is 17.8% to 2.1 A resolution. The RMS deviation from ideality is 0.010 A for bond lengths and 2.5 degrees for bond angles in both models. Three amino acid positions beneath the active site are occupied by Thr 40, Ile 55, and Ser 91 in hen, pheasant, and other avian lysozymes, and by Ser 40, Val 55, and Thr 91 in guinea fowl and American quail lysozymes. In spite of their internal location, the structural changes associated with these substitutions are small. The pheasant enzyme has an additional N-terminal glycine residue, probably resulting from an evolutionary shift in the site of cleavage of prelysozyme. In the 3-dimensional structure, this amino acid partially fills a cleft on the surface of the molecule, close to the C alpha atom of Gly 41 and absent in lysozymes from other species (which have a large side-chain residue at position 41: Gln, His, Arg, or Lys). The overall structures are similar to those of other c-type lysozymes, with the largest deviations occurring in surface loops. Comparison of the unliganded and antibody-bound models of pheasant lysozyme suggests that surface complementarity of contacting surfaces in the antigen-antibody complex is the result of local, small rearrangements in the epitope. Structural evidence based upon this and other complexes supports the notion that antigenic variation in c-type lysozymes is primarily the result of amino acid substitutions, not of gross structural changes.
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PMID:Crystal structures of pheasant and guinea fowl egg-white lysozymes. 806 8


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