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)

His 15 of hen lysozyme is located at the protein surface and is partly buried by the neighboring residues. The side chain of His 15 forms hydrogen bonds with surrounding residues and these hydrogen bonds are somewhat buried. A series of mutant lysozymes at the position 15 (Gly, Ala, Val, and Phe) was prepared, and their stabilities were analyzed by GdnHCl denaturation and X-ray crystallography. The mutants were less stable than the wild type at pH 5.5 and 35 degrees C. In H15G and H15A, X-ray crystallography revealed two fixed water molecules at the mutated region, which formed similar hydrogen bonds to those in the wild type. On the other hand, it was suggested that the hydrogen bonds were disrupted and that several unfavorable van der Waals' contacts occurred in H15V and H15F. Therefore, we concluded that His 15 stabilized the lysozyme structure by forming hydrogen bonds and the best packing with the neighboring residues. Moreover, we found that the method of protein stabilization by increasing the hydrophobicity of an amino acid residue was not always effectively applicable, especially when the residue had formed a hydrogen bond.
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PMID:Analysis of the stability of mutant lysozymes at position 15 using X-ray crystallography. 934 77

Several variants of T4 lysozyme have been identified that sequester small organic ligands in cavities or clefts. To evaluate potential binding sites for non-polar molecules, we screened a number of hydrophobic large-to-small mutants for stabilization in the presence of benzene. In addition to Leu99-->Ala, binding was indicated for at least five other mutants. Variants Met102-->Ala and Leu133-->Gly, and a crevice mutant, Phe104-->Ala, were further characterized using X-ray crystallography and thermal denaturation. As predicted from the shape of the cavity in the benzene complex, mutant Leu133-->Gly also bound p-xylene. We attempted to enlarge the cavity of the Met102-->Ala mutant into a deep crevice through an additional substitution, but the double mutant failed to bind ligands because an adjacent helix rearranged into a non-helical structure, apparently due to the loss of packing interactions. In general, the protein structure contracted slightly to reduce the volume of the void created by truncating substitutions and expanded upon binding the non-polar ligand, with shifts similar to those resulting from the mutations.A polar molecule binding site was also created by truncating Arg95 to alanine. This creates a highly complementary buried polar environment that can be utilized as a specific "receptor" for a guanidinium ion. Our results suggest that creating a deficiency through truncating mutations of buried residues generates "binding potential" for ligands with characteristics similar to the deleted side-chain. Analysis of complex and apo crystal structures of binding and non-binding mutants suggests that ligand size and shape as well as protein flexibility and complementarity are all determinants of binding. Binding at non-polar sites is governed by hydrophobicity and steric interactions and is relatively permissive. Binding at a polar site is more restrictive and requires extensive complementarity between the ligand and the site.
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PMID:Generation of ligand binding sites in T4 lysozyme by deficiency-creating substitutions. 951 55

Soft-shelled turtle egg-white lysozyme was purified and sequenced. Lysozyme was reduced and carboxymethylated to fragment it with trypsin, V8 protease and CNBr. The peptides yielded were purified by RP-HPLC and sequenced. Every trypsin peptide was overlapped by V8 protease peptides and CNBr fragment. The amino acid sequence was compared with other lysozymes. This lysozyme has an extra Gly residue at N-terminus, which was found in pheasant lysozyme. Further, this lysozyme has an insertion of a Gly residue between 47 and 48 residues when compared with chicken lysozyme, as found in human lysozyme, therefore it proved that this lysozyme has the largest number of amino acids (131 aa) in chicken type lysozymes. The amino acid substitutions were found at subsites E and F. Namely Phe34, Arg45, Thr47, and Arg114 were replaced by His, Tyr, Arg, and Tyr, respectively. The time course using N-acetylglucosamine pentamer as a substrate showed a reduction of the rate constant for glycosidic cleavage and increase of binding free energy for subsites E and F, which proved the contribution of amino acids mentioned above for substrate binding at subsites E and F.
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PMID:Reptile lysozyme: the complete amino acid sequence of soft-shelled turtle lysozyme and its activity. 953 89

In order to clarify the structural role of subsite B of hen egg-white lysozyme in hydrolytic activity towards a carbohydrate substrate, we analysed the structures of Trp-62-->Gly and Asp-101-->Gly mutant hen lysozymes, which have no side chain at positions 62 or 101, complexed with a substrate analogue, (N-acetyl-d-glucosamine)3 [(GlcNAc)3], using X-ray crystallography. The overall protein structures in the mutant lysozyme complexes were almost identical to those in the wild type. In the crystals of all the mutant complexes, the (GlcNAc)3 molecule, which is an inhibitor of wild-type lysozyme, had no inhibitory effect, but was hydrolysed as a substrate. One of the products, (GlcNAc)2, the reducing end of which is an alpha-anomer, was bound in an unproductive binding mode, protruding from the active-site cleft, and was able to act as an inhibitor. Hydrolysis of the synthetic substrate by the mutants occurred in a beta-anomer-retaining manner, and so the alpha-anomer product was converted from the beta-anomer product. Thus the interactions of Asp-101 and Trp-62 in subsite B are not essential for the catalytic mechanism, but co-operatively enhance the affinity of the substrate in the productive binding mode, other than the inhibitor in the unproductive mode.
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PMID:Structural and functional effect of Trp-62-->Gly and Asp-101-->Gly substitutions on substrate-binding modes of mutant hen egg-white lysozymes. 963 64

Achromobacter beta-lytic protease (blp), one of the bacteriolytic proteases secreted by Achromobacter lyticus, exhibited both peptidase and bacteriolytic activities at alkaline pH. The protease was strongly inhibited by 1,10-phenanthroline, and one zinc atom was detected in the molecule by ion-spray mass spectrometry. The zinc-protease specifically cleaved Gly-X bonds in peptides and possibly possessed subsites S2, S1, S1', and S2' for binding substrate [Schecter, I. and Berger, A. (1967) Biochem. Biophys. Res. Commun. 27, 157-162]. Blp lysed Staphylococcus aureus and Micrococcus luteus cells more efficiently than Achromobacter alpha-lytic protease (alp) and lysozyme, thus being responsible for the high bacteriolytic activity of A. lyticus. In the lysis of bacterial cell walls, blp hydrolyzed both the D-Ala-Gly/Ala bond at the linkage between the peptide subunit and the interpeptide and the Gly-Gly bond in the interpeptide bridge. These results indicate that blp is a highly active bacteriolytic enzyme with a broad bacteriolytic spectrum, which acts primarily by splitting the linkage between the peptide subunit and the interpeptide in the peptidoglycan.
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PMID:Bacteriolytic activity and specificity of Achromobacter beta-lytic protease. 968 23

The amino acid sequence of monal pheasant lysozyme and its activity were analyzed. Carboxymethylated lysozyme was digested with trypsin and the resulting peptides were sequenced. The established amino acid sequence had one amino acid substitution at position 102 (Arg to Gly) comparing with Indian peafowl lysozyme and four amino acid substitutions at positions 3 (Phe to Tyr), 15 (His to Leu), 41 (Gln to His), and 121 (Gln to His) with chicken lysozyme. Analysis of the time-courses of reaction using N-acetylglucosamine pentamer as a substrate showed a difference of binding free energy change (-0.4 kcal/mol) at subsites A between monal pheasant and Indian peafowl lysozyme. This was assumed to be caused by the amino acid substitution at subsite A with loss of a positive charge at position 102 (Arg102 to Gly).
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PMID:The amino acid sequence of monal pheasant lysozyme and its activity. 983 34

Two water-soluble chitosan derivatives, N-succinyl-chitosan (Suc-chi; average MW 3x10(5)) and glycol-chitosan (Gly-chi; average MW 1.5x10(5)), were examined concerning their biodisposition characteristics in order to evaluate their possible use as water-soluble drug carriers. Their body distribution and urinary excretion were investigated by i.v. administration of FITC-labeled Suc-chi (FTC-Suc-chi) and FITC-labeled Gly-chi (FTC-Gly-chi) to normal and Sarcoma 180 solid tumor-bearing mice. In normal mice, both polymers showed good retention in blood circulation; especially, FTC-Suc-chi exhibited a long half-life of 51 h, and its distribution to other tissues was very small. FTC-Gly-chi was distributed into the kidney to a relatively high extent. In tumor-bearing mice, FTC-Suc-chi and FTC-Gly-chi were eliminated faster from the blood circulation than in normal mice, that is, with half-lives of 11 and 7 h, respectively. FTC-Suc-chi was less partitioned to the tumor tissue but accumulated more easily into it compared with FTC-Gly-chi. This suggested the enhanced permeability and retention (EPR) effect of Suc-chi and explained the previous result that a water-soluble Suc-chi-mitomycin C conjugate injected intravenously exhibited a good effect against Sarcoma 180 solid tumor. FTC-Gly-chi showed greater distribution to the kidney than in normal mice. Urinary excretion studies indicated the faster excretion of both polymers in tumor-bearing mice. The molecular weight of the products excreted into urine indicated that both polymers should be pretty resistant to the hydrolytic enzyme, lysozyme. Taking toxicities into account, Suc-chi is considered to be available as a drug carrier showing long systemic retention and tumor accumulation.
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PMID:Biodisposition characteristics of N-succinyl-chitosan and glycol-chitosan in normal and tumor-bearing mice. 1007 38

The Val99-Gly 104 variable region in egg white lysozyme is part of the active site cleft and of the epitope recognized by some monoclonal antibodies. In general, this loop is found in a conformation inflected towards the active site (proximal conformational) such as in free hen lysozyme (HEL). But in a lysozyme such as Japanese quail's (JEL), the loop turns away from the active site cleft (distal conformation). In order to differentiate sequence effects from crystal packing, we generated and refined loop conformations for the 99-104 variable region in lysozyme, then estimated their relative conformational free energies. Some of the results indicate that (i) the flexibility of the 99-104 segment is much greater for HEL than for JEL sequences when unconstrained by the crystal lattice, (ii) for JEL, only distal structures are favored, while for HEL the states span the zone between proximal and distal regions, and (iii) epitopes elucidated from crystal structures may not always be conserved in solution. For the JEL loop, model building shows that an energy-costly distal to proximal transition appears necessary. Finally, analysis of available structural data indicates that changes of humidity, temperature and pressure on loop conformation are negligible.
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PMID:Molecular modeling of an active loop structure in lysozyme. Sequence effects or crystal packing? 1021 56

The mutation Glu108-->Val (E108V) in T4 lysozyme was previously isolated as a second-site revertant that specifically compensated for the loss of function associated with the destabilizing substitution Leu99-->Gly (L99G). Surprisingly, the two sites are 11 A apart, with Leu99 in the core and Glu108 on the surface of the protein. In order to better understand this result we have carried out a detailed thermodynamic, enzymatic and structural analysis of these mutant lysozymes as well as a related variant with the substitution Leu99-->Ala. It was found that E108V does increase the stability of L99G, but it also increases the stability of both the wild-type protein and L99A by essentially equal amounts. The effects of E108V on enzymatic activity are more complicated. The mutation slightly reduces the maximal rate of cell wall hydrolysis of wild-type, L99G and L99A. At the same time, L99G is an unstable protein and rapidly loses activity during the course of the assay, especially at temperatures above 20 degrees C. Thus, even though the double mutant L99G/E108V has a slightly lower maximal rate than L99G, over a period of 20-30 minutes it hydrolyzes more substrate. This decrease in the rate of thermal inactivation appears to be the basis of the action of E108V as a second-site revertant of L99G. Mutant L99A creates a cavity of volume 149 A(3). Instead of enlarging this cavity, mutant L99G results in a 4-5 A displacement of part of helix F (residues 108-113), creating a solvent-accessible declivity. In the double mutant, L99G/E108V, this helix returns to a position akin to wild-type, resulting in a cavity of volume 203 A(3). Whether the mutation Glu108-->Val is incorporated into either wild-type lysozyme, or L99A or L99G, it results in a decrease in crystallographic thermal factors, especially in the helices that include residues 99 and 108. This increase in rigidity, which appears to be due to a combination of increased hydrophobic stabilization plus a restriction of conformational fluctuation, provides a structural basis for the increase in thermostability.
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PMID:Structural analysis of a non-contiguous second-site revertant in T4 lysozyme shows that increasing the rigidity of a protein can enhance its stability. 1051 6

To minutely understand the effect of foreign N-terminal residues on the conformational stability of human lysozyme, five mutant proteins were constructed: two had Met or Ala in place of the N-terminal Lys residue (K1M and K1A, respectively), and others had one additional residue, Met, Gly or Pro, to the N-terminal Lys residue (Met(-1), Gly(-1) and Pro(-1), respectively). The thermodynamic parameters for denaturation of these mutant proteins were examined by differential scanning calorimetry and were compared with that of the wild-type protein. Three mutants with the extra residue were significantly destabilized: the changes in unfolding Gibbs energy (DeltaDeltaG) were -9.1 to -12.2 kJ.mol-1. However, the stability of two single substitutions at the N-terminal slightly decreased; the DeltaDeltaG values were only -0.5 to -2.5 kJ.mol-1. The results indicate that human lysozyme is destabilized by an expanded N-terminal residue. The crystal structural analyses of K1M, K1A and Gly(-1) revealed that the introduction of a residue at the N-terminal of human lysozyme caused the destruction of hydrogen bond networks with ordered water molecules, resulting in the destabilization of the protein.
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PMID:Effect of foreign N-terminal residues on the conformational stability of human lysozyme. 1056 12


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