Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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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 three aspartic acid residues that form part of the Ca-binding site of mares' milk lysozyme have apparent pK values of 4.9, 4.3 and 4.1. The fluorescence of tryptophan has been used to compare the denaturation of mares' milk lysozyme by guanidinium chloride at various concentrations of Ca with that of hens' egg-white lysozyme (EC 3.2.1.17) and alpha-lactalbumin. Fluorescence revealed an intermediate stage in the denaturation of mares' milk lysozyme. The Ca-free form of mares' milk lysozyme is slightly more stable than that of alpha-lactalbumin, but its interaction with Ca is similar to that of alpha-lactalbumin, since only the native state binds Ca. Three-state models of denaturation can usefully be displayed on a ternary diagram.
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PMID:Effect of calcium on the stability of mares' milk lysozyme. 140 55

The "right-sided" and "left-sided" substrate binding modes at the lower saccharide binding subsites (D-F sites) of chicken lysozyme were investigated by utilizing mutant lysozymes secreted from yeast. We constructed the following mutant lysozymes; "left-sided" substitution of Asn46 to Asp, deletion of Thr47, and insertion of Gly between Thr47 and Asp48 and "right-sided" substitution of Asn37 to Gly. Analyses of their activities and substrate binding abilities showed that Asn46 and Thr47 are involved in the initial enzyme-substrate complex and Asn37 is involved in the transition state. These results support an earlier proposal that interactions between substrate and residues at the left side of lysozyme stabilize a catalytically inactive enzyme-substrate complex, while interactions between substrate and residues at the right side stabilize the catalytically active complex [Pincus, M. R., & Scheraga, H. A. (1979) Macromolecules 12, 633-644]. These results are also consistent with the proposed kinetic mechanism for lysozyme reaction that the rearrangement of an initial enzyme-substrate complex (beta-complex) to another complex (gamma-complex) is required for catalytic hydrolysis [Banerjee S. K., Holler, E., Hess, G. P., & Rupley, J. A. (1975) J. Biol. Chem. 250, 4355-4367].
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PMID:Left-sided substrate binding of lysozyme: evidence for the involvement of asparagine-46 in the initial binding of substrate to chicken lysozyme. 142 Jan 52

From fluorescence measurements on mixtures of bis-ANS and equine lysozyme and from Ca(2+)-dependent hydrophobic interaction chromatography of equine lysozyme, it is demonstrated that Ca2+ binding induces a conformational change upon which hydrophobic regions in the protein become less accessible. Bis-ANS fluorescence titrations in the absence of Ca2+ and in 2 mM Ca2+ are also performed with equine alpha-lactalbumin variants B and C. These variants differ by an amino-acid exchange Asp----Ile at residue 95. The fluorescence titration curves indicate that the accessibility of the probe to the Ca2+ conformers is clearly influenced by the mutation. The Ca(2+)-dependent exclusion of a hydrophobic domain is used in a new and simplified method for preparing lysozyme and alpha-lactalbumins simultaneously from equine milk whey.
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PMID:Hydrophobic interaction of lysozyme and alpha-lactalbumin from equine milk whey. 150 92

Threonine 59, a helix-capping residue at the amino terminus of the longest helix in T4 phage lysozyme, was substituted with valine, alanine, glycine, serine, asparagine, and aspartic acid. The valine, alanine, and glycine replacements were observed to be somewhat more destabilizing than serine, asparagine, and aspartic acid. The crystal structures of the different variants showed that changes in conformation occurred at the site of substitution, including Asp 61, which is nearby, as well as displacement of a solvent molecule that is hydrogen-bonded to the gamma-oxygen of Thr 59 in wild-type lysozyme. Neither the structures nor the stabilities of the mutant proteins support the hypothesis of Serrano and Fersht (1989) that glycine and alanine are better helix-capping residues than valine because a smaller-sized residue allows better hydration at the end of the helix. In the aspartic acid and asparagine replacements the substituted side chains form hydrogen bonds with the end of the helix, as does threonine and serine at this position. In contrast, however, the Asp and Asn side chains also make unusually close contacts with carbon atoms in Asp 61. This suggests a structural basis for the heretofore puzzling observations that asparagine is more frequently observed as a helix-capping residue than threonine [Richardson, J. S., & Richardson, D. C. (1988) Science 240, 1648-1652] yet Thr----Asn replacements at N-cap positions in barnase were found to be destabilizing [Serrano, L., & Fersht, A. R. (1989) Nature 342, 296-299].(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Dissection of helix capping in T4 lysozyme by structural and thermodynamic analysis of six amino acid substitutions at Thr 59. 156 17

Aminoacetylation of lysine residues and the modification of arginine by 1,2-cyclohexanedione to N7,N8-(dihydroxy-1,2-cyclohexylidene)arginine were used for probing the surface topology of hen-eggwhite lysozyme as a model protein. The molecular identification of lysine and arginine modification sites was provided by molecular weight determinations of modified and unmodified tryptic peptide mixtures (peptide mapping) using 252Cf plasma desorption mass spectrometry. At conditions of limited chemical modification, mass-spectrometric peptide-mapping analyses of lysozyme derivatives enabled the direct assignment of relative reactivities of lysine and arginine residues at different reaction times and reagent concentrations. The relative reactivities of lysine residues showed a direct correlation with their surface accessibilities from x-ray structure data. For the reaction with 1,2-cyclohexanedione, a selective modification at Arg-5, -125, -112, and -73 was identified, and an inverse correlation of relative reactivities with the surface accessibility ratios of the N7- and the N8-guanidino functions was obtained. By examination of the x-ray structural data of lysozyme, this selective modification was attributed to intramolecular catalysis because of the presence of neighboring proton acceptor groups, such as the Asp-119 carboxylate group for Arg-125 and the Trp-123 and Arg-125 carbonyl groups for Arg-5.
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PMID:Protein surface topology-probing by selective chemical modification and mass spectrometric peptide mapping. 160 73

To experimentally examine the functional roles of somatically derived structural variation in the lysozyme-binding mAb HyHEL-10, we have introduced three different point mutations and one insertion at two different sites in HyHEL-10 by site-directed mutagenesis and expression of the mutant antibodies. Mutation of Asp----Ala at position 101 of the H chain returns a somatically mutated residue to its germline sequence for HyHEL-10, and reduces affinity for chicken lysozyme by approximately 9000-fold. Lengthening the third H chain hypervariable region by two amino acids reduces affinity by about 2000-fold. Two mutations, Asp----Thr at position 101 in the H chain and Lys----Thr at position 49 in the L chain, model somatic differences found in another structurally related but functionally distinguishable mAb and minimally decrease affinity for chicken lysozyme. The H chain mutation Asp101VVH----Thr has little effect on affinity for other avian lysozymes but does alter relative fine specificity for these lysozymes. The L chain mutation Lys49VK----Thr increases affinity for duck lysozyme by approximately fivefold. Neither of the positions mutated, 101 in the H chain nor 49 in the L chain, nor the residues near the insertion contact lysozyme in the x-ray structure of the HyHEL-10 F(ab)-HEL complex. The results suggest that these mutations, which model observed somatic mutations, produce functional variation by indirect or long-range effects.
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PMID:Experimental analysis by site-directed mutagenesis of somatic mutation effects on affinity and fine specificity in antibodies specific for lysozyme. 172 69

The production of a mutant hen lysozyme is described in which Asp-52, one of the catalytically important residues, is replaced by Ser. The mutant enzyme has very low catalytic activity but NMR studies show that its structure is closely similar to that of the wild-type protein. NMR experiments also show that well defined complexes are formed with GlcNAc4 and GlcNAc6 bound in the active site of the mutant enzyme. These complexes have been examined using electrospray mass spectrometry (ESMS). The most intense peaks arise from the uncomplexed protein indicating that dissociation takes place in the mass spectrometer under the conditions used here. Peaks from minor species corresponding to complexes between the protein and the oligosaccharides are, however, also observed. The possibility that the latter arise from novel covalent enzyme-saccharide complexes is discussed.
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PMID:A study of D52S hen lysozyme-GlcNAc oligosaccharide complexes by NMR spectroscopy and electrospray mass spectrometry. 173 71

To determine the energetic and structural consequences of placing a charged group within the core of a protein, two "buried charge" mutants, Met 102----Lys (M102K) and Leu 133----Asp (L133D) were constructed in phage T4 lysozyme. Both proteins fold at neutral pH, although they are substantially less stable than wild type. The activity of M102K is about 35% that of wild type, while that of L133D is about 4%. M102K could be crystallized, and its structure was determined at high resolution. The crystal structure (at pH 6.8) of the mutant is very similar to that of wild type except for the alpha-helix that includes residues 108-113. In wild-type lysozyme, one side of this helix is exposed to solvent and the other contacts Met 102. In the M102K structure this alpha-helix becomes much more mobile, possibly allowing partial access of Lys 102 to solvent. The stability of M102K, determined by monitoring the unfolding of the protein with CD, is pH-dependent, consistent with the charged form of the substituted amino acid being more destabilizing than the uncharged form. The pKa of Lys 102 was estimated to be 6.5 both by differential titration and also by NMR analysis of isotopically labeled protein with 13C incorporated at the C epsilon position of all lysines. As the pH is lowered below pH 6.5, the overall three-dimensional structure of M102K at room temperature appears to be maintained to pH 3 or so, although there is evidence for some structural adjustment possibly allowing solvent accessibility to the protonated form of Lys 102.
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PMID:Structural and thermodynamic consequences of burying a charged residue within the hydrophobic core of T4 lysozyme. 174 70

The indole C-2(delta 1) carbon of Trp 62 in hen egg-white lysozyme was selectively labeled with 13C through a series of reactions involving N'-formylkynurenine 62-lysozyme with K13CN, NaBH4-reduction, and acid-catalyzed dehydration. [delta 1-13C]Trp 62-lysozyme in which Trp 62 is labeled with 90% 13C has the same chemical and enzymatic properties as the native protein. The reverted lysozyme gave a single 13C-NMR signal at 125 ppm. pH-titration of the 13C signal indicated a transition at pH 3.9 for the free enzyme. In the presence of (GlcNAc)3, the resonance signals were shifted 0.5-1 ppm upfield, and the transitions in the titration curve were observed at pH 3.9 and 6.5. Asp 52 and Glu 35 were assigned to the groups with pKas of 3.9 and 6.5, respectively. In [2-13C]AHT 62-lysozyme, which has 3-(2-amino-3-hydroxy-3H-[2-13C]indol-3-yl)alanine (AHT) at position 62, AHT 62 behaved quite differently from Trp 62 on pH-titration of the 13C-label. These results suggest that a conformational change around Trp 62 is induced upon ionization of the catalytic residue and that the structural flexibility of the side chain of this aromatic residue in the substrate binding site is closely related to the function of lysozyme.
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PMID:Site-specific 13C-labeling of Trp 62 in hen egg-white lysozyme: preparation and 13C-NMR titration of [delta 1-13C]Trp 62-lysozyme. 176 25

Free energy simulation methods are used to analyse the effects of the mutation Arg-96----His on the stability of bacteriophage T4 lysozyme and of Ile-96----Ala on the stability of barnase. By use of thermodynamic integration, the contributions of specific interactions to the free energy change are evaluated. It is shown that a number of contributions that stabilize the wild-type or the mutant partially cancel in the overall free energy difference; some of these involve the unfolded state. Comparison of the results with conclusions based on structural and thermodynamic data leads to new insights into the origin of the stability difference between wild-type and mutant proteins. For the charged-to-charged amino acid mutation in T4 lysozyme, the importance of the contributions of more distant residues, solvent water and the covalent linkage involving the mutated amino acid are of particular interest. Also, the analysis of the Arg-96 to His mutation with respect to the interactions with the C-terminal end of a helix (residues 82-90) indicates that the nearby carbonyl groups (Tyr-88 and Asp-89) make the dominant contribution, that the amide groups do not contribute significantly and that the helix dipole model is inappropriate for this case. For the non-polar-to-non-polar amino acid mutation in barnase, the solvent contribution is unimportant, and covalent terms are shown to be significant because they do not cancel between the folded and unfolded state.
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PMID:Simulation analysis of the stability mutants R96H of bacteriophage T4 lysozyme and I96A of barnase. 181 97


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