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)

Since it has been uncertain whether residue 103 in hen egg-white lysozyme is aspartic acid or asparagine, we reexamined the identity of this residue. To avoid complication, the tryptic peptide T-13 (Ile 98-Arg 112) was further cleaved. The peptide containing residues Gly 102-Arg 112 was obtained by tryptic digestion of lysozyme modified at Asp 101 with diethylenetriamine. The peptide containing residues Ile 98-homoserine 105 was obtained by BrCN treatment of peptide T-13. Both Edman degradation of the former peptide and carboxypeptidase X digestion of the latter peptide identified residue 103 in hen egg-white lysozyme as asparagine.
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PMID:Identification of residue 103 in hen egg-white lysozyme. 730 22

The pH difference absorption spectra of human lysozyme [EC 3.2.1.17] were measured. The difference spectra in the acidic region had a peak at 300 nm, as observed for hen and turkey lysozymes. The pH dependence curve of the extinction difference at 300 nm was well interpreted in terms of the pK values of the catalytic groups (3.4 for Asp 52 and 6.8 for Glu 35 at 0.1 ionic strength and 25 degrees C) determined from the pH dependence of the circular dichroism at 303.5 nm (Kuramitsu et al. (1974) J. Biochem. 76, 671--683) and the fluorescence excited at 305 nm (Kuramitsu et al. (1978) J. Biochem. 83, 159--170). The difference spectra of human lysozyme in the alkaline pH region were characteristic of tyrosyl ionization. The perturbation of tryptophyl residues, which had been observed for hen and turkey lysozymes (Kuramitsu & Hamaguchi (1979) J. Biochem. 85, 443--456), was not observed for human lysozyme. On the basis of the pH dependence curves of the extinction difference at 245 and and 295 nm, we roughly estimated the apparent pK values of the six tyrosyl residues as 9.2 9.2, 10.5, 10.9, 12.4, and 12.5. A time-dependent spectral change observed above pH 11 was not due to the exposure of buried tyrosyl residues on alkali denaturation but was due mainly to disulfide cleavage and exposure of buried tryptophyl residues.
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PMID:Ionization of the catalytic groups and tyrosyl residues in human lysozyme. 739 Sep 61

The binding constants of 4-methylumbelliferyl-beta-glycosides of (GlcNAc)2 ((GlcNAc)2-MeU) and of (GlcNAc)3 ((GlcNAc)3-MeU) to hen lysozyme [EC 3.2.1.17] were determined by measuring changes in the fluorescence at 375 nm. It was shown that (GlcNAc)2-MeU and (GlcNAc)3-MeU bind mainly at subsites B, C, and D, and A, B, C, and D, respectively, with the terminal MeU group bound at subsite D. The rate of hydrolysis of (GlcNAc)3-MeU catalyzed by hen and turkey lysozymes was determined at 0.1 ionic strength and 42 degrees C in the pH range of 2 to 8. The release of 4-methylumbelliferone was followed fluorimetrically. The pH dependences of kcat, kcat/Km, and Km were analyzed assuming that that nonproductive binding occurs competitively and that the molecular species with ionized Asp 52 and protonated Glu 35 is active. Comparison of the pH dependences of the kinetic constants for hen lysozyme with those for turkey lysozyme, in which Asp 101 of hen lysozyme is replaced by Gly, made it possible to determine the pK values of Asp 52, Glu 35, and Asp 101. The pK values of Asp 52 and Glu 35 were 3.60 and 6.20, respectively, for hen and turkey lysozymes and 3.95 and 6.55, respectively, for their nonproductive complexes. The pK value of Asp 101 of hen lysozyme was 4.20 for the free enzyme, 3.30 for the nonproductive complex, and 3.95 for the productive complex. These pK values, except for the pK value of Asp 52 of the nonproductive complex, are in excellent agreement with those determined by spectroscopic methods in our laboratory (Kurasmitsu et al. (1974) J. Biochem. 76, 671--683; (1975) ibid. 77, 291--301; (1977) ibid. 82, 585--597; (1978) ibid. 83, 159--170). This demonstrates that lysozyme-catalyzed hydrolysis can be fully explained in terms of the proposal based on the X-ray data (Blake et al. (1967) Proc. Roy. Soc. B167, 378--388), with regard to the participation of Asp 52 and Glu 35.
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PMID:Hydrolysis of 4-methylumbelliferyl N-acetyl-chitotrioside catalyzed by hen and turkey lysozymes. pH dependence of the kinetics constants. 739 Sep 76

The interactions of beta-methyl-GlcNAc, (GlcNAc)2, and (GlcNAc)3 with hen egg-white lysozyme [EC 3.2.1.17] in which Trp 62 is modified to kynurenine (Kyn 62-lysozyme) were studied by measuring the changes in the CD band and fluorescence due to the kynurenine at various pH values. The pH profiles of the binding constants of these saccharides to Kyn 62-lysozyme were very similar to those to intact lysozyme, although the binding constants were lower for the modified lysozyme than for intact lysozyme. The pK values of Asp 52, Glu 35, Asp 48, and Asp 101 in Kyn 62-lysozyme and in its complexes with beta-methyl-GlcNAc and with (GlcNAc)2 were in agreement with those of intact lysozyme and its complexes. The pK values of Asp 52 and Glu 35 in the modified lysozyme-(GlcNAc)3 complex were also in agreement with those of the complex with intact lysozyme, but the pK shift of Asp 101 was smaller for Kyn 62-lysozyme than for intact lysozyme. The significance of the decreased binding constants to Kyn 62-lysozyme is discussed. The pH dependence of the CD band due to the kynureinine in Kyn 62-lysozyme was interpreted in terms of the participation of the catalytic groups, Asp 52 (apparent pK 3.5) and Glu 35 (apparent pK 6.0), and the amino group of the kynurenine (apparent pK 0.75). This indicates that the ionization of the catalytic groups affects the state around Trp 62 and supports the previous proposal that there is a relation between the state around Trp 62 and the ionization of Glu 35 (Ikeda, K. & Hamaguchi, K. (1973) J. Biochem. 74, 221--230; (1975) ibid. 77, 1--16; Nakae et al. (1975) J. Biochem. 77, 993--1006). The intrinsic pK value of the amino group of the kynurenine at position 62 shifted from 2.1 to 0.8 on complexing with (GlcNAc)3, indicating between the kynurenine and the sugar residue at subsite B.
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PMID:Binding of substrate analogs to hen lysozyme in which Trp 62 is modified to kynurenine. 739 Sep 77

Binding of 4-methylumbelliferyl chitotetraoside ((GlcNAc)4-MeU) to hen lysozyme [EC 3.2.1.17] was studied by measuring changes in fluorescence at 375 nm. Hydrolysis of (GlcNAc)4-MeU catalyzed by lysozyme was studied by measuring the release of 4-methylumbelliferone from (GlcNAc)4-MeU fluorimetrically, and the kinetic constants were determined in the pH range of 2 to 8 at 0.1 ionic strength and 42 degrees C. The binding and kinetic data showed that (GlcNAc)4-MeU binds to subsites A to E (productive binding) and subsites A to D with the nonreducing sugar residue extending beyond subsite A (nonproductive binding). The fraction of the productive complex was 0.77 at pH 8.5. The pH dependence of the kinetic constants was analyzed assuming that the molecular species with ionized Asp 52 and protonated Glu 35 is active and Asp 101 participates in the binding (Phillips (1966) Sci. Am. 215, 78-90; Blake et al. (1967) Proc. Roy. Soc. B167, 378-388), and the pK values of these groups were determined. The pK values of Asp 52, Glu 35, and Asp 101 were 3.60, 6.20, and 4.20, respectively, for free lysozyme, 3.40, 6.55, and 3.40, respectively, for the productive complex, and 3.95, 6.55, and 3.30, respectively, for the nonproductive complex. The pK values for free lysozyme were in excellent agreement with those obtained by analysis of the kinetic constants for (GlcNAc)3-MeU (Yang & Hamaguchi (1980) J. Biochem. 87, 1003-1014). The free energy of activation was 24 kcal mol-1 at pH 5.2. Comparison with the corresponding value obtained for hydrolysis of (GlcNAc)6 (Banerjee et all (1975) J. Biol. Chem. 250, 4355-4367) suggests that the interactions of GlcNAc residues with subsites E and F in the transition state are important in lysozyme catalysis. Hydrolysis of (GlcNAc)2-MeU catalyzed by lysozyme was also studied, and the Kcat/Km values for (GlcNAc)2-MeU, (GlcNAc)3-MeU, and (GlcNAc)4-MeU were compared.
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PMID:Hydrolysis of 4-methylumbelliferyl N-acetyl-chitotetraoside catalyzed by hen lysozyme. 741 25

To examine the effect of a conformational constraint introduced into the Arg-Gly-Asp (RGD) sequence on cell adhesion activity, we have constructed mutant proteins by inserting RGD-containing sequences flanked by two Cys residues between Val74 and Asn75 of human lysozyme. CRGDC-, CRGDSC-, and CGRGDSC-inserted mutant lysozymes were expressed in yeast, purified, and designated as Cys-RGD3, Cys-RGD4, and Cys-RGD5, respectively. In baby hamster kidney cells, these mutants were shown to possess high cell adhesion activity by interaction with vitronectin receptor (integrin alpha v beta 3), and this activity is 2-3-fold higher than that of the RGDS-inserted mutant lysozyme, RGD4. The mutant proteins also inhibited the binding of human fibrinogen to its receptor (integrin alpha IIb beta 3) at a lower concentration than the RGD4 protein. Peptide mapping and mass spectrometric analyses showed that the two inserted Cys residues in these mutants are linked to each other without any effects on the mode of the four disulfide bonds present in native human lysozyme. These results suggest that the introduction of a conformational constraint into the RGD region significantly increases the cell adhesion activity. The conformation of the RGD region in Cys-RGD4 was modeled by a Monte Carlo simulation. Most of the sampled conformations were grouped into three classes; the first is characterized by an extended Gly conformation, the second assumes a type II' beta turn, and the third has a salt bridge between Arg and Asp.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Functional analysis and modeling of a conformationally constrained Arg-Gly-Asp sequence inserted into human lysozyme. 752 52

The cell-adhesive protein Cys-RGD4 has been constructed using a yeast expression system by inserting the sequence Cys-Arg-Gly-Asp-Ser-Cys (CRGDSC) between Val74 and Asn75 of human lysozyme [Yamada, T., Uyeda, A., Kidera, A. & Kikuchi, M. (1994b) Biochemistry 33, 11678-11683]. The Cys74a, Arg74b, Gly74c, Asp74d, Ser74e, Cys74f-lysozyme mutant, purified from the yeast culture supernatant contained glycosylated variants, in addition to the unglycosylated form. Peptide mapping analyses suggested that the glycosylation occurred at the Thr70 residue in the Cys-RGD4 molecule. Electrospray ionization mass spectrometric analysis demonstrated the presence of two hexose residues in the major variant, and one, three, four, or five hexose residues in the minor variants. All of these hexose residues were identified as mannose by analysis of the oligosaccharide mixture obtained by mild alkaline treatment of the variants. No other glycosylation was observed, although the Cys-RGD4 molecule possesses a total of 12 threonine and serine residues. In addition, the Thr70 residue is not glycosylated in either native lysozyme or the Arg-Gly-Asp-Ser (RGDS)-inserted mutant, RGD4 [Yamada, T., Matsushima, M., Inaka, K., Ohkubo, T., Uyeda, A., Maeda, T., Titani, K., Sekiguchi, K. & Kikuchi, M. (1993) J. Biol. Chem. 268, 10588-10592]. Thus, this O-glycosylation seems to be specific for both the mutant lysozyme molecule and the site of the threonine residue. Structural analyses of these lysozymes by X-ray crystallography suggest that the conformation of the serine-containing or threonine-containing region can affect the specificity of yeast O-glycosylation.
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PMID:O-glycosylation of the Thr70 residue of cell-adhesive lysozyme in yeast. 760 Nov 60

The effects of additives on the nonenzymatic deamidation of an Asn residue in a peptide and racemization of Asp and/or Asn in lysozyme were investigated at pH 6 and 100 degrees C. These chemical reactions were accelerated by the addition of phosphate ions. Several salts suppressed the deamidation in the presence of phosphate ions, while the salts did not affect the deamidation in the absence of phosphate ion at pH 6 and 100 degrees C. The results indicated that the effect of the salts was due to the suppression of phosphate catalysis. On the other hand, trifluoroethanol (TFE), which induces the conversion of random coiled polypeptides to secondary structured ones, dramatically suppressed the deamidation of an Asn residue in a peptide. The rate of deamidation in the presence of TFE was comparable to that of asparagine (free amino acid), which was very slowly deamidated. Because TFE could not suppress the deamidation of free asparagine, the suppression of the deamidation of an Asn residue in a peptide was attributed to suppression of the catalysis by the peptide bond in the carboxyl terminus. Since the inactivation of lysozyme was caused by multiple chemical reactions such as the deamidation and racemization, it was expected that the inactivation of lysozyme could be prevented by the addition of salts or TFE. Thus, it was confirmed that salts and TFE suppressed the lysozyme inactivation at pH 6 and 100 degrees C.
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PMID:Stabilization of lysozyme against irreversible inactivation by suppression of chemical reactions. 762 34

This paper describes a general method to calculate the pKas of ionizable groups in proteins. Electrostatic calculations are carried out using the finite difference Poisson-Boltzmann (FDPB) method. A formal treatment of the calculation of pKas within the framework of the FDPB method is presented. The major change with respect to previous work is the specific incorporation of the complete charge distribution of both the neutral and charged forms of each ionizable group into the formalism. This is extremely important for the treatment of salt bridges. A hybrid statistical mechanical/Tanford-Roxby method, which is found to be significantly faster than previous treatments, is also introduced. This simplifies the problem of summing over the large number of possible ionization states for a complex polyion. Applications to BPTI and serine proteases suggest that the calculations can be quite reliable. However, the necessity of including bound waters in the treatment of the Asp-70... His-31 salt bridge in T4 lysozyme and experience with other proteins suggest that additional factors ultimately need to be considered in a comprehensive treatment of pKas in proteins.
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PMID:On the calculation of pKas in proteins. 768 Dec 10

The complex formed between hen egg white lysozyme (HEL) and the monoclonal antibody HyHEL-10 Fab fragment has an interface composed of van der Waals interactions, hydrogen bonds, and a single ion pair. The antibody overlaps part of the active site cleft. Putative critical residues within the epitope region of HEL, identified from the x-ray crystallographic structure of the complex, were replaced by site-directed mutagenesis to probe their relative importance in determining affinity of the antibody for HEL. Twenty single mutations of HEL at three contact residues (Arg-21HEL, Asp-101HEL, and Gly-102HEL) and at a partially buried residue (Asn-19HEL) in the epitope were made, and the effects on the free energies of dissociation were measured. A correlation between increased amino acid side-chain volume and reduced affinity for HELs with mutations at position 101 was observed. The D101GHEL mutant is bound to HyHEL-10 as tightly as wild-type enzyme, but the delta delta Gdissoc is increased by about 2.2 kcal (9.2 kJ)/mol for the larger residues in this position. HEL variants with lysine or histidine replacements for arginine at position 21 are bound 1.4-2.7 times more tightly than those with neutral or negatively charged amino acids in this position. These exhibit 1/40 the affinity for HyHEL-10 Fab compared with wild type. There is no side-chain volume correlation with delta delta Gdissoc at position 21. Although Gly-102HEL and Asn-19HEL are in the epitope, replacements at these positions have no effect on the affinity of HEL for the antibody.
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PMID:High-resolution mapping of the HyHEL-10 epitope of chicken lysozyme by site-directed mutagenesis. 768 15


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