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)

An acidic 1,2-alpha-mannosidase from fungus, Aspergillus saitoi (now designated Aspergillus phoenicis), is highly specific for 1,2-alpha-mannosidic linkage in the high-mannose type oligosaccharide at pH 5.0. The predicted amino acid sequence of several peptide regions, including aspartic acid and glutamic acid, bears striking similarities to 1,2-alpha-mannosidases from fungi, yeast and mouse. Active site determination of the enzyme expressed in Saccharomyces cerevisiae cells was performed by site-directed mutagenesis. Substitutions of Asp-269 to Glu and of the Glu-residues, Glu-273, Glu-411, Glu-414 and Glu-474, to Asp altered the drastic decrease of specific activities with Man alpha 1-2Man-OMe and Man9-GlcNAc2-PA as substrates and shifted the optimal pH of the mutant enzymes. From the present results, Asp-269 is probably in the ionized COO- form, whereas one of four glutamic acid residues, probably Glu-411, is the un-ionized COOH form according to the analogy of a plausible mechanism for lysozyme catalysis. It is assumed that three glutamic acid residues, Glu-273, Glu-414, and Glu-474, are probably binding sites of substrate.
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PMID:Five crucial carboxyl residues of 1,2-alpha-mannosidase from Aspergillus saitoi (A. phoenicis), a food microorganism, are identified by site-directed mutagenesis. 932 67

We have determined the structure of murine MHC class II I-Ak in complex with a naturally processed peptide from hen egg lysozyme (HEL residues 50-62) at 1.9 A resolution. These results provide a structural basis for the I-Ak peptide-binding motif. Binding is established by the deep burial of five anchor side chains into specific pockets of the I-Ak binding groove, with a zen-like fit of an aspartic acid in the P1 pocket. We also show that in the I-Ak alpha chain, a bulge occurs in the first strand of the peptide-binding platform, an insertion probably common to all I-A and HLA-DQ alleles. The I-Ak beta chain has a deletion in the helical region adjacent to the P7 pocket and an insertion in the helical region neighboring the P1 pocket. As a result of these structural features, the extended HEL peptide dips low into the center of the I-Ak groove and reaches toward solvent at its C-terminal end.
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PMID:Crystal structure of I-Ak in complex with a dominant epitope of lysozyme. 952 48

Chicken egg white lysozyme is progressively inhibited by diazoacetyl-DL-norleucine methyl ester (DANME) and by chlorambucil at concentrations of 3.4 x 10(-3) M and 5 x 10(-3) M respectively over a three-hour time period. DANME inhibits lysozyme activity to the extent of 87%, and chlorambucil inhibits the enzyme to the extent of 93%. N,N',N"-triacetylchitotriose [(NAG)3], which binds to subsites A, B and C of the enzyme protects lysozyme from DANME inhibition to the extent of 40% of the total activity when added to the enzyme at a concentration of 3.6 x 10(-3) M prior to the addition of DANME. (NAG)3 protects the enzyme from inhibition by chlorambucil to the extent of 14% of the total activity when added to the enzyme at a concentration of 5.6 x 10(-3) M prior to the addition of chlorambucil. Since DANME reacts exclusively with carboxyl groups, and since aspartic acid 101 is required for binding the carbohydrate substrate at site A, it is suggested that (NAG)3 may bind reversibly to the active site of the enzyme, thereby protecting aspartic acid 101 from esterification by DANME and subsequent inactivation. Chlorambucil, which may react with carboxyl, amino, imidazole and thiol groups, more likely acts upon a larger number of susceptible sites, thereby causing irreversible alkylation and conformation changes. As a bifunctional alkylating agent, it may also cross-link with two available nucleophiles. The K(m) for lysozyme with M. lysodeikticus as a substrate in wholly aqueous medium was determined to be 0.05 mg/mL. The inhibitor exhibits a partially uncompetitive upon pre-incubation with the enzyme, and a mixed inhibition between competitive and noncompetitive when pre-incubated with the substrate.
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PMID:The effect of chlorambucil upon lysozyme activity. 1002 96

The binding of murine monoclonal antibody HyHEL-5 to lysozyme has been the subject of extensive crystallographic, computational, and experimental investigations. The complex of HyHEL-5 with hen egg lysozyme (HEL) features salt bridges between Fab heavy chain residue Glu(50), and Arg(45) and Arg(68) of HEL. This interaction has been predicted to play a dominant role in the association on the basis of molecular electrostatics calculations. The association of aspartic acid and glutamine mutants at position 50(H) of the cloned HyHEL-5 Fab with HEL and bobwhite quail lysozyme (BQL), an avian variant bearing an Arg(68) --> Lys substitution in the epitope, was characterized by isothermal titration calorimetry and sedimentation equilibrium. Affinities for HEL were reduced by 400-fold (E50(H)D) and 40,000-fold (E50(H)Q) (DeltaDeltaG degrees estimated at 4.0 and 6.4 kcal mol(-1), respectively). The same mutations reduce affinity for BQL by only 7- and 55-fold, respectively, indicating a reduced specificity for HEL. The loss of affinity upon mutation is in each case primarily due to an unfavorable change in the enthalpy of the interaction; the entropic contribution is virtually unchanged. An enthalpy-entropy compensation exists for each interaction; DeltaH degrees decreases, while DeltaS degrees increases with temperature. The DeltaCp for each mutant interaction is less negative than the wild-type. Mutant-cycle analysis suggests the mutations present in the HyHEL-5 Fab mutants are linked to those present in the BQL with coupling energies between 3 and 4 kcal mol(-1).
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PMID:Salt links dominate affinity of antibody HyHEL-5 for lysozyme through enthalpic contributions. 1048 Aug 91

The three-dimensional structure of the lytic transglycosylase from bacteriophage lambda, also known as bacteriophage lambda lysozyme, complexed to the hexasaccharide inhibitor, hexa-N-acetylchitohexaose, has been determined by X-ray crystallography at 2.6 A resolution. The unit cell contains two molecules of the lytic transglycosylase with two hexasaccharides bound. Each enzyme molecule is found to interact with four N-acetylglucosamine units from one hexasaccharide (subsites A-D) and two N-acetylglucosamine units from the second hexasaccharide (subsites E and F), resulting in all six subsites of the active site of this enzyme being filled. This crystallographic structure, therefore, represents the first example of a lysozyme in which all subsites are occupied, and detailed protein-oligosaccharide interactions are now available for this bacteriophage lytic transglycosylase. Examination of the active site furthermore reveals that of the two residues that have been implicated in the reaction mechanism of most other c-type lysozymes (Glu35 and Asp52 in hen egg white lysozyme), only a homologous Glu residue is present. The lambda lytic transglycosylase is therefore functionally closely related to the Escherichia coli Slt70 and Slt35 lytic transglycosylases and goose egg white lysozyme which also lack the catalytic aspartic acid.
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PMID:Crystal structure of the lytic transglycosylase from bacteriophage lambda in complex with hexa-N-acetylchitohexaose. 1134 31

Compartmentalization of enzymes in the nanometric-scaled container, to improve their stability and availability, has recently attracted a strong interest in the field of pharmaceutics. In this study, the enzymatic activity of lysozyme in the core of polyion complex (PIC) micelles, which were formed from egg white lysozyme and poly(ethylene glycol)-poly(alpha,beta-aspartic acid) block copolymer (PEG-P(Asp)), was evaluated using a colorimetric method. Apparent enzymatic activity of lysozyme entrapped in the core of PIC micelles remarkably increased compared to that of free lysozyme, which is mainly attributed to a decrease in the observed Michaelis constant (K(m,obs)). The reciprocal of the K(m,obs) values nicely correlated to the corona thickness of PIC micelles, suggesting that the corona layer of PIC micelle may act as the reservoir of the substrate, p-nitrophenyl penta-N-acetyl-beta-chitopentaoside. This result indicates that the enzymatic activity can be controlled by changing the corona thickness of PIC micelles through a variation in the mixing ratio of PEG-P(Asp) to lysozyme. This type of PIC micelle system entrapping enzyme in the core might be useful for the design of diagnostic as well as targetable therapeutic systems of enzyme including antibody-directed enzyme prodrug therapy (ADEPT).
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PMID:Pronounced activity of enzymes through the incorporation into the core of polyion complex micelles made from charged block copolymers. 1138 87

A catalytically inactive mutant of hen egg white lysozyme was constructed by site-directed mutagenesis to elucidate the role of enzymatic activity on its antimicrobial activity against Gram-positive bacteria. The catalytic residue aspartic acid at position 52 of lysozyme was substituted with serine (D52S-Lz) and the mutant cDNA was inserted into a yeast expression vector, pYES-2. Western blot analysis indicated that the mutation did not affect secretion of the D52S-Lz lysozyme into the medium of the expressing Saccharomyces cerevisiae, INVSC1. In addition, circular dichroism and fluorescence spectral analysis revealed no change in the structure of D52S-Lz compared to that of wild-type (Wt-Lz) lysozyme. The mutation (D52S) abolished the catalytic activity of lysozyme. Antimicrobial tests against Staphylococcus aureus and Bacillus subtilis revealed that the catalytically inactive D52S-Lz was as bactericidal as the Wt-Lz lysozyme. Heat treatment leading to enzyme inactivation had no effect on the bactericidal activity of either wild-type or the mutant D52S-Lz lysozyme. The binding affinity of D52S-Lz to the isolated peptidoglycan of S. aureus was unaffected. Our results provide the first demonstration of direct genetic evidence that the antimicrobial activity of lysozyme is operationally independent of its muramidase activity, and strongly suggest the antimicrobial action of lysozyme is due to structural factors.
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PMID:Genetic evidence that antibacterial activity of lysozyme is independent of its catalytic function. 1159 65

We have analyzed a panel of T cell hybridomas specific for the chemically dominant epitope of hen egg-white lysozyme 48-61 which has asparagine 59 as an important T cell receptor contact residue. A number of T cells recognize 48-61 with asparagine at position 59, but not the aspartic acid or isoaspartic acid derivatives. Conversely, we find T cells that specifically recognize 48-61 bearing an isoaspartic acid at residue 59, but not asparagine. For other T cells, asparagine, aspartic acid, or isoaspartic acid at residue 59 is irrelevant. We present evidence that our previous distinction between type A and type B T cells is not explained by asparagine deamidation at residue 59.
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PMID:Deamidation of asparagine in a major histocompatibility complex-bound peptide affects T cell recognition but does not explain type B reactivity. 1160 44

To better understand the relation between sequence and structure, and in an attempt to simplify the protein folding problem, a series of alanine substitutions was introduced into bacteriophage T4 lysozyme. In contrast to previous studies in this system, which were restricted to single alpha-helices, the present analysis included a helix-turn-helix region, a loop-helix region, and two alpha-helices that were well separated in the three-dimensional structure. It was shown previously that T4 lysozyme is very tolerant of alanine substitutions within alpha-helices, especially at solvent-exposed sites. The present study shows that the protein is also tolerant of such substitutions in turn and loop regions, although less than in helices. The results confirm that the structural information in the amino acid sequence is highly redundant. For example, the protein with the sequence 127AAAAAALAAAAWAAA141 folds normally, has melting temperature only 0.8 degrees C lower than wildtype, and has a crystal structure that is also very similar to wildtype. Polyalanine substitutions within turns or loops can, however, lead to differences in structure and in folding. In one example the triple substitution K35A/S36A/P37A caused this region of the molecule to change to a more helical conformation. In a second case the mutant with the sequence 34AAAAALAAAKAALAAA49, which spans a loop-helix region, had a dramatically altered thermal unfolding transition, suggesting that this region may tend to form a single, uninterrupted, helix. Substitution of Ala38 in the above construct with aspartic acid caused the unfolding to be more like wildtype, suggesting that residue 38, which is at a helix-capping position in the wildtype structure, provides an initiation signal that is essential in the polyalanine mutant for the correct formation of alpha-helix 39-50. In a typical protein, the information that codes for the 3D structure is presumably distributed over many amino acids. The present results suggest that in simplified sequences the key folding information may be restricted to a subset of critical residues, and so be more readily accessible to experimental analysis.
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PMID:A helix initiation signal in T4 lysozyme identified by polyalanine mutagenesis. 1248 88

Shockman, Gerald D. (Temple University School of Medicine, Philadelphia, Pa.), Joseph J. Kolb, Bohdan Bakay, Margaret J. Conover, and Gerrit Toennies. Protoplast membrane of Streptococcus faecalis. J. Bacteriol. 85:168-176. 1963.-The membrane fraction of Streptococcus faecalis (ATCC 9790) was isolated and purified, by a variety of procedures, from cultures that were grown under closely controlled conditions of physiological age and nutrition. The most satisfactory method required the use of lysozyme-to-cell ratios below 0.01 and the intermediate formation of protoplasts in osmotically protective media. Amino acid analyses of three of the membrane preparations indicated a characteristic and constant, but not unusual, pattern; 42% of the membranes from threonine-depleted and 49 to 55% of the membranes from log-phase cultures were accounted for as protein. Significant quantities of d-alanine or d-aspartic acid were not detected, indicating the absence of contaminating cell-wall substance. Essentially, all of the nitrogen was accounted for as amino acids. The lipid content of membranes from stationary-phase threonine-depleted (36%) and valine-depleted (40%) cultures was significantly higher than the corresponding fraction of exponential-phase cultures (28%). The phosphorus content of the membrane lipid was relatively constant (2.8 to 3.0%), and the nitrogen content was extremely low (0.12 to 0.26%). Thus, changes in the composition of the membrane fraction occurred during the transition of log-phase cells into threonine- or valine-depleted cells.
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PMID:Protoplast membrane of Streptococcus faecalis. 1398 37


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