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 peptidoglycans of Moraxella glucidolytica and Moraxella lwoffi grown on aliphatic hydrocarbons were isolated. They contained muramic acid, glucosamine, alanine, D-glutamic acid and mesodiaminoimelic acid in a molar ratio of about 0.5:0.5:1.6:1.0:1.0 (M. glucidolytica) and 0.8:0.7:1.3:1.0:1.0 (M. lwoffi). The peptidoglycans were lysozyme-resistant. However, when treated with formanide, they could be partially degraded by lysozyme. The fragments were purified and their structure determined. In both strains, the peptide subunits consisted mainly of tripeptides (L-Ala-D-Glu-meso-DAP) and tetrapeptides (L-Ala-D-glu-meso-DAP-D-Ala), most of them being directly cross-linked. It is concluded that in both strains the primary structures of the peptidoglycans are closely related.
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PMID:Structure of the peptidoglycans of Moraxella glucidolytica and Moraxella lwoffi grown on hydrocarbons. 87 Dec 29

Dicarboxylic amino acids constitute the most numerous residues of insoluble elastin in which are potentially ionizable in the physiological range of pH. These residues are essential in facilitating productive electrostatic interaction between elastase and elastin. The present study has investigated the possibility that the glutamic and aspartic acid residues of elastin are amidated. Acid-labile amide-bound ammonia of elastin was quantitated after hydrolysis of the insoluble protein with 2 M HC1 by incubating aliquots of microdistilled hydrolysates with glutamate dehydrogenase, excess alpha-ketoglutarate, and reduced nicotinamide adenine dinucleotide and measuring the resultant decrease in A340 due to oxidation of the dinucleotide cofactor. It was found that ligament elastin purified by repeated autoclaving contains approximately 2.29 mumol of acid-labile amide nitrogen per 10 mg of protein, a value equivalent to approximately 70% of the total number of dicarboxylic amino acid residues. Independent analysis of the amide content was obtained by amino acid analysis of an esterified and reduced elastin sample in which the free dicarboxylic amino acid residues had been converted to the corresponding alcohol derivatives. This analysis indicated that autoclaved ligament elastin contains approximately 18 glutamine, 3 asparagine, 4 glutamic acid and 5 aspartic acid residues per 1000 residues, in good agreement with the analysis of total acid-labile ammonia. The esterified and reduced elastin derivative was nearly inert as an elastase substrate, consistent with a lack of free dicarboxylic amino acid residues. However, addition of sodium dodecyl sulfate to this elastin derivative restores enzyme-substrate charge complementarity, and the elastin-ligand complex was readily hydrolyzed by elastase at the fully stimulated rate, emphasizing the control such ligands can exert in elastolysis. The amide bonds of elastin were found to be significantly more resistant to hydrolysis by 0.1 M NaOH at 98 degrees C than were those of lysozyme or free amidated amino acids. The finding that most of dicarboxylic amino acid residues of elastin exist at neutral amides further emphasizes the apolar character of elastin and has bearing upon the metabolic susceptibility, ligand-binding ability and structural aspects of this connective tissue protein.
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PMID:Amidated carboxyl groups in elastin. 93 66

Studies on the structure and substrate specificity of purified rat kidney nuclear (RKN) lysozyme are reported. The carboxyl and amino terminal residues of RKN-lysozyme were found to be leucine and alanine respectively. The amino acid composition indicated similarities and differences as compared with that of hen egg white (HEW) lysozyme. There were alterations in the nine amino acid residues, Lys, His, Arg, Asp, Glu, Pro, 1/2 Cys, Tyr and Trp. The other nine residues were present in identical proportions to those of HEW-lysozyme. The decrease in the arginine and aspartic acid residues was found to be compensated by the increase in the number of lysine, histidine and glutamic acid residues. The overall ratio of the acidic to basic amino acids has thus been conserved in the mammalian enzyme. In addition, RKN-lysozyme contained decreased numbers of Trp, Tyr and 1/2 Cys, and increased numbers of proline residues as found in HEW-lysozyme. RKN-lysozyme did not cross react with heterologous antibodies produced against HEW-lysozyme, and vice versa. RKN-lysozyme showed distinct specificity towards the lysis of M. luteus. Against this substrate, it was three times more efficient than HEW-lysozyme. It also cleaved E. coli B, but its efficiency was half as much as with M. luteus. However, it cleaved P. septica and B. subtilis at a rate similar to HEW-lysozyme under identical conditions.
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PMID:Structure-activity studies on mammalian tissue lytic enzymes: chemical characterization and substrate specificity of rat kidney nuclear lysozyme. 95 82

Trp108 of chicken lysozyme is in van der Waals contact with Glu35, one of two catalytic carboxyl groups. The role of Trp108 in lysozyme function and stability was investigated by using mutant lysozymes secreted from yeast. By the replacement of Trp108 with less hydrophobic residues, Tyr (W108Y lysozyme) and Gln (W108Q lysozyme), the activity, saccharide binding ability, stability, and pKa of Glu35 were all decreased with a decrease in the hydrophobicity of residue 108. Namely, at pH 5.5 and 40 degrees C, the activities of W108Y and W108Q lysozymes against glycol chitin were 17.3 and 1.6% of that of wild-type lysozyme, and their dissociation constants for the binding of a trimer of N-acetyl-D-glucosamine were 7.4 and 309 times larger than that of wild-type lysozyme, respectively. For the reversible unfolding at pH 3.5 and 30 degrees C, W108Y and W108Q lysozymes were less stable than wild-type lysozyme by 1.4 and 3.6 kcal/mol, respectively. As for the pKa of Glu35, the values for W108Y and W108Q lysozymes were found to be lower than that for wild-type lysozyme by 0.2 and by 0.6 pKa unit, respectively. The pKa of Glu35 in lysozyme was also decreased from 6.1 to 5.4 by the presence of 1-3 M guanidine hydrochloride, or to 5.5 by the substitution of Asn for Asp52, another catalytic carboxyl group. Thus, both the hydrophobicity of Trp108 and the electrostatic interaction with Asp52 are equally responsible for the abnormally high pKa (6.1) of Glu35, compared with that (4.4) of a normal glutamic acid residue.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Multiple role of hydrophobicity of tryptophan-108 in chicken lysozyme: structural stability, saccharide binding ability, and abnormal pKa of glutamic acid-35. 161 Jul 99

The role of aspartic acid 53 of human lysozyme (peptidoglycan N-acetylmuramoylhydrolase, EC 3.2.1.17) has been investigated by a site-directed mutagenesis. In order to clarify the importance of precise positioning of the negatively charged carboxylate group in the active site geometry, both the three-dimensional structure and the enzymatic function of glutamic acid 53 human lysozyme (Glu-53 human lysozyme) have been characterized in comparison with those of wild type enzyme. Glu-53 human lysozyme was crystallized and analysed by X-ray crystallography. No remarkable difference in the conformation of whole molecule except the side chain of 53rd residue was observed. In spite of full retention of the binding activities against either beta-1,4-linked trisaccharide of N-acetylglucosamine ((GlcNAc)3) or the corresponding hexasaccharide ((GlcNAc)6), the conversion of Asp-53 to Glu reduced the enzymatic activities against both bacterial cell substrate and p-nitrophenyl penta-N-acetyl-beta(1----4)-chitopentaoside (p-NO2-(GlcNAc)5) to a few percent of the activities of wild type enzyme. Calculation of electrostatic potential around the reaction center predicted that no significant change in pKa of Glu-35 was caused by the mutation. These results indicate that the precise positioning of the negatively charged carboxylate in the geometry of reaction center is essential for the rate enhancement in the catalytic action of lysozyme, and suggest that Asp-53 of human lysozyme participates in the catalytic action not simply in an electrostatical manner but partly in a nucleophilical manner.
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PMID:The importance of precise positioning of negatively charged carboxylate in the catalytic action of human lysozyme. 191 46

Bacteriophage T4 lysozyme is a basic molecule with an isoelectric point above 9.0, and an excess of nine positive charges at neutral pH. It might be expected that it would be energetically costly to bring these out-of-balance charges from the extended, unfolded, form of the protein into the compact folded state. To determine the contribution of such long-range electrostatic interactions to the stability of the protein, five positively charged surface residues, Lys16, Arg119, Lys135, Lys147 and Arg154, were individually replaced with glutamic acid. Eight selected double, triple and quadruple mutants were also constructed so as to sequentially reduce the out-of-balance formal charge on the molecule from +9 to +1 units. Each of the five single variant proteins was crystallized and high-resolution X-ray analysis confirmed that each mutant structure was, in general, very similar to the wild-type. In the case of R154E, however, the Arg154 to Glu replacement caused a rearrangement in which Asp127 replaced Glu128 as the capping residue of a nearby alpha-helix. The thermal stabilities of all 13 variant proteins were found to be fairly similar, ranging from 0.5 kcal/mol more stable than wild-type to 1.7 kcal/mol less stable than wild-type. In the case of the five single charge-change variants, for which the structures were determined, the changes in stability can be rationalized in terms of changes in local interactions at the site of the replacement. There is no evidence that the reduction in the out-of-balance charge on the molecule increases the stability of the folded relative to the unfolded form, either at pH 2.8 or at pH 5.3. This indicates that long-range electrostatic interactions between the substituted amino acid residues and other charged groups on the surface of the molecule are weak or non-existent. Furthermore, the relative stabilities of the multiple charge replacement mutant proteins were found to be almost exactly equal to the sums of the relative stabilities of the constituent single mutant proteins. This also clearly indicates that the electrostatic interactions between the replaced charges are negligibly small. The activities of the charge-change mutant lysozymes, as measured by the rate of hydrolysis of cell wall suspensions, are essentially equal to that of the wild-type lysozyme, but on a lysoplate assay the mutant enzymes appear to have higher activity.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Cumulative site-directed charge-change replacements in bacteriophage T4 lysozyme suggest that long-range electrostatic interactions contribute little to protein stability. 194 34

femA is a chromosomally encoded factor, occurring naturally in Staphylococcus aureus, which is essential for the expression of high-level methicillin resistance in this organism. The production of a low-affinity penicillin-binding protein, PBP2a or PBP2', which is intimately involved with methicillin resistance in S. aureus, is not influenced by femA. To elucidate a possible physiological function of the 48-kDa protein encoded by femA, several related methicillin-resistant, methicillin-susceptible, and Tn551 insertionally inactivated femA mutants were analyzed for possible changes in cell wall structure and metabolism. Independent of the presence of mec, the methicillin resistance determinant, all femA mutants had a reduced peptidoglycan (PG) glycine content (up to 60% in the molar ratio of glycine/glutamic acid) compared to that of related femA+ parent strains. Additional effects of femA inactivation and the subsequent decrease in PG-associated glycine were (i) reduced digestion of PG by recombinant lysostaphin, (ii) unaltered digestion of PG by Chalaropsis B-muramidase, (iii) reduced cell wall turnover, (iv) reduced whole-cell autolysis, and (v) increased sensitivity towards beta-lactam antibiotics. Also, the PG-associated glycine content of a femA::Tn551 methicillin-susceptible strain was restored concomitantly with the methicillin resistance to a level almost equal to that of its femA+ methicillin-resistant parent strain by introduction of plasmid pBBB31, encoding femA.
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PMID:femA, which encodes a factor essential for expression of methicillin resistance, affects glycine content of peptidoglycan in methicillin-resistant and methicillin-susceptible Staphylococcus aureus strains. 204 71

Polypeptide hormone signal transmission by receptor tyrosine kinases requires the rapid reversal of tyrosine phosphorylation by protein phosphotyrosine phosphatases (PPTPases). We studied hepatic PPTPases in the rat with emphasis on acute and chronic regulation by insulin. PPTPase activity with artificial substrates ([32P]Tyr-reduced, carboxyamidomethylated, and maleylated lysozyme and [32P]Tyr-poly[glutamic acid:tyrosine] 4:1) was present in distinct membrane, cytoskeletal, and cytosolic fractions. These PPTPase activities were unaffected by alloxan diabetes. Acute administration of insulin to normal animals also did not change PPTPase activity in liver plasma membranes or endosomal membranes. Although alloxan diabetes did not affect PPTPase activity measured with artificial substrates or with epidermal growth factor receptors, a decrease in insulin receptor dephosphorylation was noted. Dephosphorylation of hepatic receptors from normal and diabetic rats by membrane PPTPase from control rats was similar. These results indicate that alloxan diabetes does not lead to a generalized effect on hepatic PPTPase activity, although a substrate-specific decrease in activity with the insulin receptor may occur.
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PMID:Hepatic protein phosphotyrosine phosphatase. Dephosphorylation of insulin and epidermal growth factor receptors in normal and alloxan diabetic rats. 216 29

The complete amino-acid sequence of pig alpha-lactalbumin has been determined. It was obtained by microsequencing of the native protein and the peptides derived after tryptic or cyanogen bromide cleavage. The tryptic peptides were separated by a rapid microbore HPLC method. Pig alpha-lactalbumin is 122 amino acids long and differs from the bovine homologue by 26 exchanged residues. Of the two prolines present in bovine alpha-lactalbumin, one has been deleted in the pig structure. All previously sequenced alpha-lactalbumins have shown glutamic acid at position 49, which is known to be the active site in the homologous lysozyme c structure. This residue is replaced by phenylalanine in pig alpha-lactalbumin indicating that the pig protein is the first alpha-lactalbumin with complete loss of all lysozyme functional residues.
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PMID:The complete primary structure of alpha-lactalbumin isolated from pig (Sus scrofa) milk. 222 64

A full-length Caenorhabditis elegans cDNA that encodes the alpha subunit of casein kinase II was inserted into the inducible bacterial expression vector pET3a to generate the plasmid pCK alpha. Escherichia coli DE21 lysozyme S that was transformed with pCK alpha expressed soluble, catalytically active casein kinase II alpha upon induction with isopropyl beta-D-thiogalactopyranoside. The expressed alpha subunit was purified to homogeneity with a 60% yield by chromatography on CM-Sephadex, P-11 phosphocellulose, and heparin-agarose. The Mr values estimated from sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Mr = 42,000) or calculated from hydrodynamic measurements (s20,w = 3.3 S, Stokes radius = 2.8 nm, Mr = 37,000) were similar, thereby indicating that the expressed enzyme is monomeric. The native holoenzyme and the expressed alpha subunit exhibited several similar properties including the utilization of both ATP and GTP as substrates and the susceptibility to inhibition of phosphotransferase activity by low concentrations of heparin. However, the kcat for E. coli-derived alpha was only 9% of the kcat for the native holoenzyme, and catalytic activity was not stimulated by polyamines. Recombinant casein kinase II alpha aggregates at low ionic strength, and the aggregation is partially reversible. A mutant alpha subunit in which Lys74 and Lys75 were substituted by glutamic acid residues was constructed by site-directed mutagenesis. The mutant enzyme was not inhibited by typically effective concentrations of heparin (e.g. IC50 = 0.3 micrograms/ml) because the affinity of modified recombinant casein kinase II Glu-74Glu-75 for heparin decreased approximately 70-fold. Thus, Lys74 and Lys75 are implicated in the heparin binding, inhibitory domain. The successful expression of casein kinase II alpha in E. coli will facilitate the analysis of the structural basis for functional domains in this enzyme.
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PMID:Expression of wild-type and mutated forms of the catalytic (alpha) subunit of Caenorhabditis elegans casein kinase II in Escherichia coli. 224 6


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