pH dependence of the binding constants of N-acetylglucosamine monomers to hen and turkey egg-white lysozymes.

The binding constants of N-acetylglucosamine (G1cNAc) and its methyl alpha- and beta- glycosides to hen and turkey egg-white lysozymes [EC 3.2.1.17], in the latter of which Asp 101 is replaced by Gly, were determined at various pH values by measuring changes in the circular dichroic (DC) band at 295 nm. The binding of beta-methyl-G1cNAc to turkey and hen lysozymes perturbed the pK value of Glu 35 from 6.0 to 6.5, the pK value of Asp 52 from 3.5 to 3.9, and the pK value of Asp 66 from 1.3 to 0.7. In addition, perturbation of the pK value of Asp 101 from 4.4 to 4.0 was observed in the binding of this saccharide to hen lysozyme. The binding of alpha-methyl-GlcNAc to hen and turkey lysozymes perturbed the pK value of Glu 35 to the alkaline side by about 0.5 pH unit, the pK value of Asp 66 to the acidic side by about 0.5 pH unit, and the pK value (4.4) of an ionizable group to the acidic side by about 0.6 pH unit. The last ionizable group was tentatively assigned to Asp 48. The pK value of Asp 52 was not perturbed by the binding of this saccharide. The pH dependence curves for the binding of GlcNAc to hen and turkey lysozymes were very similar and it was suggested that Asp 48, in addition to Asp 66, Asp 52, and Glu 35, is perturbed by the binding of GlcNAc.     

Effects on tryptophyl absorption of the ionization of the catalytic carboxyls in hen and turkey lysozymes.

The difference spectra of hen and turkey egg-white lysozymes [EC 3.2.1.17] produced by acidification were measured. The difference spectra of both lysozymes had peaks at 295 and 301 nm which are characteristic of tryptophyl residues. The pH dependence curves of the extinction differences (delta eplision) at 301 nm and 295 nm for hen lysozyme were identical with the corresponding curves for turkey lysozyme. The pH dependence of delta eplision at 301 nm was analyzed assuming that the extinction at 301 nm is due to Trp 108 only, which interacts with the catalytic carboxyls, Glu 35 and Asp 52. The macroscopic pK values of Glu 35 and Asp 52 in both lysozymes thus determined were 6.0 and 3.3, respectively. These values were in excellent agreement with those determined by measuring the pH dependence of the circular dichroic band at 305 nm (Kuramitsu et al. (1974) J. Biochem, 76, 671-683; (1975) ibid. 77, 291-301). The pH dependence of delta eplision at 295 nm could not be completely explained in terms of the electrostatic effects of the catalytic groups on Trp 108.     

Measurement of the individual pKa values of acidic residues of hen and turkey lysozymes by two-dimensional 1H NMR.

The pH dependence of the two-dimensional 1H nuclear magnetic resonance spectra of hen and turkey egg-white lysozymes has been recorded over the pH range 1-7. By monitoring the chemical shifts of the resonances of the various protons of ionizable residues, individual pKa values for the acidic residues have been determined for both proteins. The pKa values are displaced, with the exception of those of the residues in the active site cleft, by an average of 1 unit to low pH compared to model compounds.     

Difference absorption spectra, circular dichroism, and disulfide cleavage of hen and turkey lysozymes in the alkaline pH region.

The difference absorption spectra of hen and turkey lysozymes in the alkaline pH region had three maxima at around 245, 292, and 300 nm and had no isosbestic points. The ratio of the extinction difference at 245 nm to that at 295 nm changed with pH. These spectral features are quite different from those observed when only tyrosyl residues are ionized, and it was impossible to determine precisely the pK values of the tyrosyl residues in lysozyme by spectrophotometric titration. A time-dependent spectral change was observed above about pH 12. This is not due to exposure of a buried tyrosyl residue on alkali denaturation. The disulfide bonds and the peptide bonds in the lysozyme molecule were cleaved by alkali above about pH 11. The intrinsic pK value of Tyr 23 of hen lysozyme was determined to be 10.24 (apparent pK 9.8) at 0.1 ionic strength and 25 degrees C from the CD titration data. Comparison of the CD titration of turkey lysozyme with that of hen lysozyme suggested that Tyr 3 and Tyr 23 in turkey lysozyme have apparent pK values of 11.9 and 9.8, respectively.     

Crystal structures of K33 mutant hen lysozymes with enhanced activities

Using random mutagenesis, we previously obtained K33N mutant lysozyme that showed a large lytic halo on the plate coating Micrococcus luteus. In order to examine the effects of mutation of K33N on enzyme activity, we prepared K33N and K33A mutant lysozymes from yeast. It was found that the activities of both the mutant lysozymes were higher than those of the wild-type lysozyme based on the results of the activity measurements against M. luteus (lytic activity) and glycol chitin. Moreover, 3D structures of K33N and K33A mutant lysozyme were solved by X-ray crystallographic analyses. The side chain of K33 in the wild-type lysozyme hydrogen bonded with N37 involved in the substrate-binding region, and the orientation of the side chain of N37 in K33 mutant lysozymes were different in the wild-type lysozyme. These results suggest that the enhancement of activity in K33N mutant lysozyme was due to an alteration in the orientation of the side chain of N37. On the other hand, K33N lysozyme was less stable than the wild-type lysozyme. Lysozyme may sacrifice its enzyme activity to acquire the conformational stability at position 33.     

Inhibition of amyloid fibrillization of hen egg-white lysozymes by rifampicin and p-benzoquinone

It has been reported that more than 20 different human proteins can fold abnormally, resulting in the formation of pathological deposits and several lethal degenerative diseases. Despite extensive investigations on amyloid fibril formation, the detailed molecular mechanism remained rather elusive. The current research, utilizing hen egg-white lysozymes as a model system, is aimed at exploring inhibitory activities of two potential molecules against lysozyme fibril formation. We first demonstrated that the formation of lysozyme amyloid fibrils at pH 2.0 was markedly enhanced by the presence of agitation in comparison with its quiescent counterpart. Next, via numerous spectroscopic techniques and transmission electron microscopy, our results revealed that the inhibition of lysozyme amyloid formation by either rifampicin or its analogue p-benzoquinone followed a concentration-dependent fashion. Furthermore, while both inhibitors were shown to acquire an anti-aggregating and a disaggregating activity, rifampicin, in comparison with p-benzoquinone, served as a more effective inhibitor against in vitro amyloid fibrillogenesis of lysozyme. It is our belief that the data reported in this work will not only reinforce the findings validated by others that rifampicin and p-benzoquinone serve as two promising preventive molecules against amyloid fibrillogenesis, but also shed light on a rational design of effective therapeutics for amyloidogenic diseases.     

Endocrine changes in the incubating and brooding turkey hen

Turkey hens were allowed to incubate eggs and to hatch and rear young. Plasma prolactin (Prl) levels increased prior to the start of continuous incubation and rose sharply as incubation progressed to reach a peak of 1178.2 +/- 221.8 ng/ml (mean +/- SEM) just before hatching. Prl levels then fell precipitously before the hens left the nest, and returned to preincubation levels (36.8 +/- 3.4 ng/ml) by the time the poults were 2 weeks old. These results show that the high plasma concentrations of Prl found during incubation are not initiated or maintained only by the stimulus of nesting. We suggest that the decline in Prl levels at the end of incubation could be related to the pipping and hatching of eggs, and the consequent shift to maternal behavior. Plasma growth hormone (GH) levels were significantly increased in hens which were brooding poults, but not in hens incubating eggs. An elevenfold, 1-day increase in plasma GH was observed immediately after the hens left the nests. Mean plasma GH levels rose from 12.0 +/- 4.7 ng/ml on the day that the hens left the nests to 133.0 +/- 32.0 ng/ml on the following day, and then declined to 23.1 +/- 9.6 ng/ml after an additional day. There were no significant changes in plasma thyroxine levels during laying, incubation and brooding. Plasma glucose concentration was significantly depressed during incubation.     

Inhibition of herpes simplex virus-induced cytopathic effect by modified hen egg-white lysozymes

Hen egg-white lysozyme and three of its basic derivatives obtained by chemical modification were tested for their activity in vitro against a wild strain of herpes simplex virus type 1. Marked inhibition of the cytopathic effect was exhibited by the three chemical derivatives and the heat-inactivated lysozyme, whereas the native enzyme displayed only modest anticytopathic activity. Enzymatic activity did not appear to be necessary for the antiherpes activity of the lysozyme compounds. Instead, other properties such as their basic nature seemed to be relevant to their antiherpes effectiveness in vitro. At the concentrations used, all compounds but one had no significant effect on cell viability and growth. Some of the compounds tested caused formation of deposits on the surface of the cells. Some correlation between deposit formation and antiherpes cytopathic activity was found. The antiherpes efficacy in vitro and toxicity of the modified lysozymes were compared with those of known antiviral agents. The lysozymes were less toxic than the reference antiviral agents, and some of them were also more active.     

Interactions on 3-deoxy and 6-deoxy derivatives of N-acetyl-D-glucosamine with hen lysozyme.

The interactions of deoxy derivatives of GlcNAc, 6-deoxy-GlcNAc, and 3-deoxy-GlcNAc with hen egg-white lysozyme [EC 3.2.1.17] were studied at various pH's by measuring the changes in the circular dichroic (CD) band at 295 nm. It was shown that 6-deoxy-GlcNAc and 3-deoxy-GlcNAc bind at subsite C of lysozyme and compete with GlcNAc. The pH dependence of the binding constant of 6-deoxy-GlcNAc was the same as that of GlcNAc. On the other hand, the binding constants of 3-deoxy-GlcNAc were 3--10 times smaller than those of GlcNAc in the pH range from 3 to 9. X-ray crystallographic studies show that O(6) and O(3) of GlcNAc at subsite C are hydrogen-bonded to the indole NH's of Trp 62 and Trp 63, respectively, but the above results indicate that Trp 63, not Trp 62, is important for the interaction of GlcNAc with lysozyme.     

Mechanism of lysozyme catalysis: role of ground-state strain in subsite D in hen egg-white and human lysozymes.

The association constants for the binding of various saccharides to hen egg-white lysozyme and human lysozyme have been measured by fluorescence titration. Among these are the oligosaccharides GlcNAc-beta(1 leads to 4)-MurNAc-beta(1 leads to 4)-GlcNAc-beta(1 leads to 4)-GlcNAc, GlcNAc-beta(1 leads to 4)-MurNAc-beta(1 leads to 4)-GlcNAc-beta(1 leads to 4)-N-acetyl-D-xylosamine, and GlcNAc-beta(1 leads to 4-GlcNAc-beta(1 leads to 4)-MurNAc, prepared here for the first time. The binding constants for saccharides which must have N-acetylmuramic acid, N-acetyl-D-glucosamine, or N-acetyl-D-xylosamine bound in subsite D indicate that there is no strain involved in the binding of N-acetyl-D-glycosamine in this site, and that the lactyl group of N-acetylmuramic acid (rather than the hydroxymethyl group) is responsible for the apparent strain previously reported for binding at this subsite. For hen egg-white lysozyme, the dependence of saccharide binding on pH or on a saturating concentration of Gd(III) suggests that the conformation of several of the complexes are different from one another and from that proposed for a productive complex. This is supported by fluorescence difference spectra of the various hen egg-white lysozyme-saccharide complexes. Human lysozyme binds most saccharides studied more weakly than the hen egg-white enzyme, but binds GlcNAc-beta(1 leads to 4)-MurNAc-beta(1leads to 4)-GlcNAc-beta(1 leads to 4)-MurNAc more strongly. It is suggested that subsite C of the human enzyme is "looser" than the equivalent site in the hen egg enzyme, so that the rearrangement of a saccharide in this subsite in response to introduction of an N-acetylmuramic acid residue into subsite D destabilizes the saccharide complexes of human lysozyme less than it does the corresponding hen egg-white lysozyme complexes. This difference and the differences in the fluorescence difference spectra of hen egg-white lysozyme and human lysozyme are ascribed mainly to the replacement of Trp-62 in hen egg-white lysozyme by Tyr-63 in the human enzyme. The implications of our findings for the assumption of superposition and additivity of energies of binding in individual subsites, and for the estimation of the role of strain in lysozyme catalysis, are discussed.     

Disulfide content of reduced hen egg white and human milk lysozymes during the folding process

In order to obtain a better understanding of the possible influence of the primary sequence of a protein on its folding pathway, renaturation of reduced human milk lysozyme was compared to that of reduced hen egg white lysozyme. Following disulfide bond formation, under identical conditions, similar products were found during the folding of both lysozymes, but the kinetics of appearance and disappearance of these intermediates as well as the appearance of the native conformation were different.     

Stomach lysozymes of ruminants. II. Amino acid sequence of cow lysozyme 2 and immunological comparisons with other lysozymes

The complete sequence of 129 amino acids has been determined for one of three closely related lysozymes c purified from cow stomach mucosa. The sequence differs from those known for 17 other lysozymes c at 39-60 positions, at one of which there has been a deletion of 1 amino acid. The glutamate replacement at position 101 and the deletion of proline at position 102 eliminate the aspartyl-prolyl bond that is present between these positions in all other mammalian lysozymes c tested. This bond appears to be the most acid-sensitive one in such lysozymes at physiological temperature. Of the 40 positions previously found to be invariant among lysozymes c, only one has undergone substitution in the cow lineage. This modest number of changes at novel positions is consistent with the inference, based on tree analysis and antigenic comparisons, that the tempo of evolutionary change in the cow lysozyme lineage has not been radically different from that in other lysozyme c lineages. The mutations responsible for the distinctive catalytic properties and stability of cow lysozyme c could be a minor fraction of the total that have been fixed in the cow lineage.