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)

1. The reactivity of alpha-chymotrypsin toward p-nitrophenylacetate has been studied in dimethylformamide, dimethylsulfoxide, formamide and methylacetamide. p-Nitrophenol is liberated in dimethylsulfoxide only. 2. The reactions of alpha-chymotrypsin in dimethylsulfoxide are characterized by the same kinetic and equilibrium constants with either the p-nitrophenyl esters of straight chain carboxylic acids (from acetic to n-caprylic) or with the "specific substrate", N-carbobenzoxy-DL-phenylalanine p-nitrophenyl ester. This signifies that reactions of alpha-chymotrypsin in dimethylsulfoxide, unlike those in aqueous medium, have no specificity toward su-strate structure. 3. The stoichiometry of alpha-chymotrypsin reactions in dimethylsulfoxide was shown to be about five moles of substrate per mole of enzyme. After attaining this stoichiometry, the reaction is completed. 4. Optical rotatory dispersion spectra indicate that in non-aqueous media alpha-chymotrypsin undergoes a large conformational transition which results in a random coil. 5. Chymotrypsinogen, trypsin, trysinogen, lysozyme and serum albumin react with p-nitrophenylacetate in dimethylsulfoxide at rates which are approximately equal to those of alpha-chymotrypsin. Thus, the "activity" of alpha-chymotrypsin in dimethylsulfoxide toward p-nitrophenylacetate does not differ from the "activity" of other proteins, some of which are not even hydrolytic enzymes.
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PMID:The reactions of alpha-chymotrypsin and related proteins with ester substrates in non-aqueous solvents. 120 14

The functional role of tyrosine-63 in the catalytic action of human lysozyme (EC 3.2.1.17) has been probed by site-directed mutagenesis. In order to identify the role of Tyr63 in the interaction with substrate, both the three-dimensional structures and the enzymatic functions of the mutants, in which Tyr63 was converted to phenylalanine, tryptophan, leucine, or alanine, have been characterized in comparison with those of the wild-type enzyme. X-ray crystallographical analysis of the mutant enzyme at not less than 1.77-A resolution indicated no remarkable change in tertiary structure except the side chain of 63rd residue. The conversion of Tyr63 to Phe or Trp did not change the enzymatic properties against the noncharged substrate (or substrate analogs) largely, while the conversion to Leu or Ala markedly reduced the catalytic activity to a few percent of wild-type enzyme. Kinetic analysis using p-nitrophenyl penta-N-acetyl-beta-(1----4)-chitopentaoside (PNP-(GlcNAc)5) as a substrate revealed that the reduction of activity should mainly be attributed to the reduction of affinity between enzyme and substrate. The apparent contribution of the phenolic hydroxyl group and the phenol group in the side chain of Tyr63 was estimated to 0.4 +/- 0.4 and 2.5 +/- 0.8 kcal mol-1, respectively. The result suggested that the direct contact between the planar side-chain group of Tyr63 and the sugar residue at subsite B is a major determinant of binding specificity toward a electrostatically neutral substrate in the catalytic action of human lysozyme.
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PMID:Dissection of the functional role of structural elements of tyrosine-63 in the catalytic action of human lysozyme. 139 Jul 8

We studied the effect of adenosine nucleotides on several aspects of the functional activation of human peripheral blood polymorphonuclear leukocytes (PMN). Radiolabeled ATP bound to PMN in a manner suggesting the existence of specific binding sites because: 1) binding was reversed (92 +/- 6%) by 100-fold excess concentrations of unlabeled ATP but minimally by either ADP (43 +/- 12%) or GTP (37 +/- 8%); and 2) binding saturation was achieved (i.e., specific binding did not increase) above 250 microM ATP. Binding studies revealed that significant ATP hydrolysis occurred, even at low temperatures and in the presence of phosphatase inhibitors. Adenosine nucleotides activated signal transduction mechanisms in PMN because: 1) 1 to 100 microM ATP and 5'-adenylylimidodiphosphate (AMP-PNP) stimulated increased production of 1,2-diacylglycerols; 2) ATP (0.5 to 500 microM) and ADP (0.1 to 10 mM) induced increased insoluble protein kinase (PKC) activity in a dose-dependent manner when used at concentrations greater than 50 microM; 3) ATP (greater than or equal to 50 microM) induced a shift in the solubility of phorbol receptors from mostly soluble (89% in untreated cells) to mostly insoluble (68%), whereas ADP, GTP, and GDP were effective at higher concentrations; and 4) greater than or equal to 50 microM ATP stimulated increased phosphorylation of endogenous PMN proteins. AMP-PNP induced PKC activity and phosphoprotein changes that were qualitatively similar to those observed when PMN were treated with ATP, suggesting that extracellular ATP hydrolysis is not required for signal transduction to activate PKC. Functionally, ATP stimulated the secretion of specific (but not azurophil) granules because vitamin B12-binding protein and low levels of lysozyme, but not beta-glucuronidase, were released; qualitatively similar results were obtained by using AMP-PNP. These results suggest that certain adenosine nucleotides employed at physiologically relevant concentrations stimulate increased 1,2-diacylglycerol production, PKC activity, granule secretion, and endogenous phosphoprotein formation in a manner that is independent of extracellular ATP hydrolysis.
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PMID:Extracellular adenosine nucleotides stimulate protein kinase C activity and human neutrophil activation. 215 72

p-Nitrophenyl beta-glycosides of N-acetylchitooligosaccharides (PNP-(GlcNAc)n n = 3-5) were examined as substrates for lysozyme [EC 3.2.1.17]. The enzyme released predominantly p-nitrophenyl N-acetyl-beta-D-glucosaminide (PNP-GlcNAc) from each substrate. Furthermore, the initial rate of PNP-GlcNAc formation in lysozyme-catalyzed hydrolysis of p-nitrophenyl penta-N-acetyl-beta-chitopentaoside (PNP-(GlcNAc)5) was about 350 and 25 times faster than those of p-nitrophenyl tri-N-acetyl-beta-chitotrioside (PNP-(GlcNAc)3) and p-nitrophenyl tetra-N-acetyl-beta-chitotetraoside (PNP-(GlcNAc)4), respectively. From these results, a new colorimetric assay method of lysozyme using PNP-(GlcNAc)5 as a substrate was developed on the basis of the determination of p-nitrophenol liberated from the substrate by lysozyme through a coupled reaction involving beta-N-acetylhexosaminidase (NAHase). The assay system gave a linear dose-response curve in the range of 2-120 micrograms of lysozyme in a 15-60 min incubation. The present assay was not significantly influenced by the ionic strength of the medium and was reproducible. This method using PNP-(GlcNAc)5 as a substrate was shown to be useful for lysozyme assay.
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PMID:p-nitrophenyl penta-N-acetyl-beta-chitopentaoside as a novel synthetic substrate for the colorimetric assay of lysozyme. 297 99

Transchitooligosylation from (GlcNAc)5 to the 4-position of PNP-GlcNAc was efficiently induced through lysozyme catalysis in an aqueous solution containing methanol with a high concentration. Use of the aqueous methanol system in this reaction not only guaranteed solubility of PNP-GlcNAc substrate, but also resulted in a remarkable increase in PNP-(GlcNAc)5 production. PNP-(GlcNAc)5 was substrate for lysozyme assay compared with PNP-(GlcNAc)4.
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PMID:Enzymatic synthesis of p-nitrophenyl N,N',N'',N'',N''''-pentaacetyl-beta-chitopentaoside in water-methanol system; significance as a substrate for lysozyme assay. 334 87

Human lysozyme dimers were prepared by the intermolecular cross-linking of the monomer that contained the mutation of either Arg41 to Cys or Ala73 to Cys with a divalent maleimide compound. Among the three kinds of possible dimers only R41C-R41C dimer, in which the two catalytic clefts can come close to each other due to the proximity of the conjugation site to the active sites, turned out to be 2.3 times more specific to a polymer substrate, ethylene glycol chitin, as compared to an oligomer substrate, PNP-(GlcNAc)5. The result indicates that it is possible to alter the substrate specificity of an enzyme by artificially controlling the orientation of the active sites.
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PMID:Alteration of the substrate specificity of human lysozyme by site-specific intermolecular cross-linking. 798 87

We showed previously that the interaction of an alphabeta heterodimeric intermediate with GroEL/GroES is essential for efficient alpha(2)beta(2) assembly of human mitochondrial branched-chain alpha-ketoacid dehydrogenase. In the present study, we further characterized the mode of interaction between the chaperonins and the native-like alphabeta heterodimer. The alphabeta heterodimer, as an intact entity, was found to bind to GroEL at a 1:1 stoichiometry with a K(D) of 1.1 x 10(-)(7) m. The 1:1 molar ratio of the GroEL-alphabeta complex was confirmed by the ability of the complex to bind a stoichiometric amount of denatured lysozyme in the trans cavity. Surprisingly, in the presence of Mg-ADP, GroES was able to cap the GroEL-alphabeta complex in cis, despite the size of 86 kDa of the heterodimer (with a His(6) tag and a linker). Incubation of the GroEL-alphabeta complex with Mg-ATP, but not AMP-PNP, resulted in the release of alpha monomers. In the presence of Mg-ATP, the beta subunit was also released but was unable to assemble with the alpha subunit, and rebound to GroEL. The apparent differential subunit release from GroEL is explained, in part, by the significantly higher binding affinity of the beta subunit (K(D) < 4.15 x 10(-9)m) than the alpha (K(D) = 1.6 x 10(-8)m) for GroEL. Incubation of the GroEL-alphabeta complex with Mg-ATP and GroES resulted in dissociation and discharge of both the alpha and beta subunits from GroEL. The beta subunit upon binding to GroEL underwent further folding in the cis cavity sequestered by GroES. This step rendered the beta subunit competent for reassociation with the soluble alpha subunit to produce a new heterodimer. We propose that this mechanism is responsible for the iterative annealing of the kinetically trapped heterodimeric intermediate, leading to an efficient alpha(2)beta(2) assembly of human branched-chain alpha-ketoacid dehydrogenase.
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PMID:Interactions of GroEL/GroES with a heterodimeric intermediate during alpha 2beta 2 assembly of mitochondrial branched-chain alpha-ketoacid dehydrogenase. cis capping of the native-like 86-kDa intermediate by GroES. 1076 84

To clarify the sorting mechanism of the lysosomal/granular proteoglycan serglycin, we treated human promonocytic U937 cells with p-nitrophenyl-beta-D-xyloside (PNP-xyl) and cycloheximide. In the absence of protein synthesis, the carbohydrate moiety of serglycin was synthesized as PNP-xyl-chondroitin sulfate (CS), and most of it was delivered to lysosomes and degraded. Further, an augmented lysosomal targeting of serglycin in the presence of tunicamycin suggested that a sorting/lectin receptor with multiple specificity was involved with an increased capacity for serglycin in the absence of N-glycosylation. Correspondingly, the cation-independent mannose 6-phosphate receptor (CI-MPR) and sortilin were observed to bind to immobilized CS. These receptors were eluted in the presence of 200-400 mM and 100-250 mM NaCl, respectively. After treating the cells with a cross-linking reagent, a portion of the sulfated proteoglycan was coimmunoprecipitated with the CI-MPR but not with sortilin. In the presence of phorbol ester, lysosomal targeting of serglycin and to a lesser extent, of cathepsin D was inhibited. We conclude that the CI-MPR participates in lysosomal and granular targeting of serglycin and basic proteins such as lysozyme associated with the proteoglycan in hematopoietic cells.
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PMID:The cation-independent mannose 6-phosphate receptor is involved in lysosomal delivery of serglycin. 1721 Jun 18

In the catalysis of sugar hydrolysis by hen egg-white lysozyme, Asp52 is thought to stabilize the reaction intermediate. This residue is involved in the well-ordered hydrogen bonding network including Asn46, Asp48, Ser50 and Asn59 on the anti-parallel beta-sheet, designated as a 'platform', on which the substrate sugar sits. To reveal the role of this hydrogen bonding network in the hydrolysis, we characterized Asn59 mutants by biochemical and crystallographic studies. Surprisingly, the introduction of only a methylene group by the Asn59Gln mutation markedly reduced the bacteriolytic activity and abolished the hydrolytic activity towards the synthetic substrate, PNP-(GlcNAc)(5). A similar result was also obtained with the Asn59Asp mutant. The crystal structure of the Asn59Asp mutant in complex with the substrate analogue revealed that, as in the wild-type, the (GlcNAc)(3) was bound in the A-B-C subsites. The reduced activity would be caused by subtle changes in the side-chain orientations as well as the electrostatic characteristics of Asp59, resulting in the rearrangement of the hydrogen bonding network of the platform. These results suggest that the precise locations of these 'platform' residues, maintained by the well-ordered hydrogen bonding network, are crucial for efficient hydrolysis.
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PMID:Importance of the hydrogen bonding network including Asp52 for catalysis, as revealed by Asn59 mutant hen egg-white lysozymes. 1960 65

Bio-nanomaterials fabricated using a bioinspired templating technique represent a novel class of composite materials with diverse applications in biomedical, electronic devices, drug delivery, and catalysis. In this study, Au nanoparticles (NPs) are synthesized within the solvent channels of cross-linked lysozyme crystals (CLLCs) in situ without the introduction of extra chemical reagents or physical treatments. The as-prepared AuNPs-in-protein crystal hybrid materials are characterized by light microscopy, transmission electron microscopy, x-ray diffraction, and Fourier-transform infrared spectroscopy analyses. Small AuNPs with narrow size distribution reveal the restriction effects of the porous structure in the lysozyme crystals. These composite materials are proven to be active heterogeneous catalysts for the reduction of 4-nitrophenol to 4-aminophenol. These catalysts can be easily recovered and reused at least 20 times because of the physical stability and macro-dimension of CLLCs. This work is the first to use CLLCs as a solid biotemplate for the preparation of recyclable high-performance catalysts.
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PMID:Cross-linked lysozyme crystal templated synthesis of Au nanoparticles as high-performance recyclable catalysts. 2368 Sep 24


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