The role of Arg114 at subsites E and F in reactions catalyzed by hen egg-white lysozyme

To understand better the role of subsites E and F in lysozyme-catalyzed reactions, mutant enzymes, in which Arg114, located on the right side of subsites E and F in hen egg-white lysozyme (HEL), was replaced with Lys, His, or Ala, were prepared. Replacement of Arg114 with His or Ala decreased hydrolytic activity toward an artificial substrate, glycol chitin, while replacement with Lys had little effect. Kinetic analysis with the substrate N-acetylglucosamine pentamer, (GlcNAc)(5), revealed that the replacement for the Arg residue reduced the binding free energies of E-F sites and the rate constant of transglycosylation. The rate constant of transglycosylation for R114A was about half of that for the wild-type enzyme. (1)H-NMR analysis of R114H and R114A indicated that the structural changes induced by the mutations were not restricted to the region surrounding Arg114, but rather extended to the aromatic side chains of Phe34 and Trp123, of which the signals are connected with each other through nuclear Overhauser effect (NOE) in the wild-type. We speculate that such a conformational change causes differences in substrate and acceptor binding at subsites E and F, lowering the efficiency of glycosyl transfer reaction of lysozyme.     

Binding of substrate analogues to subsites D, E, and F of hen egg-white lysozyme.

The pH dependence of the binding of dye, Beibrich Scarlet, to hen egg-white lysozyme[EC 3.2.1.17] was studied at ionic strength 0.3 and 25 degrees by following circular dichroic (CD)bands originating from the bound dye. This binding involved one of the catalytic groups, Glu 35. The effect of the binding of N-acetylglucosamine (GlcNAc), its dimer or trimer on the binding of this dye was also studied at pH 7.5 by measuring changes in the CD bands of the dye bound to lysozyme. It was shown that there are two sites for simultaneous binding of these saccharides in the lysozyme molecule. The stronger binding of the saccharide was noncompetitive and the weaker binding was competitive with dye binding. The binding constants for the stronger binding site (the upper portion of lysozyme cleft) were in good agreement with those previously determined by following changes in the tryptophyl CD bands of lysozyme. The binding constants to the weaker site were about 1.1 x 10(-4), 5 x 10(2), and 5M(-1) for the trimer, dimer, and monomer of GlcNAc, respectively. Assuming that the trimer, dimer, and monomer occupy subsites D, E, and F; E and F; and E, respectively, the unitary free energies of saccharide binding were estimated to be about --1.9, --3.3, and --2.7 kcal/mole for D, E, and F, respectively.     

Amino acid residues in subsites e and f responsible for the characteristic enzymatic activity of duck egg-white lysozyme

We analyzed the enzymatic properties of duck egg-white lysozyme II (DEL), which differs from hen egg-white lysozyme (HEL) in nineteen amino acid substitutions. A substrate binding study showed that DEL binds to the substrate analog at subsites A-C in the same manner as HEL. However, the experimental time-courses of DEL against the substrate N-acetylglucosamine pentamer, (GlcNAc)(5), revealed remarkably enhanced production of (GlcNAc)(2) and reduced production of (GlcNAc)(1) as compared to in the case of HEL. Computer simulation of the DEL-catalyzed reaction suggested that the amino acid substitutions at subsites E and F (Phe34 to Tyr and Asn37 to Ser) caused the great alteration in the time-courses of DEL. Subsequently, the enzymatic reactions of mutants, in which Phe34 and Asn37 in HEL were converted to Tyr and Ser, respectively, were characterized. The time-courses of the F34Y mutant exhibited profiles similar to those of HEL. In contrast, the characteristics of the N37S mutant were different from those of HEL and rather similar to those of DEL; the order of the amounts of (GlcNAc)(1) and (GlcNAc)(2) was reversed in comparison with in the case of HEL. Enhanced production of (GlcNAc)(2) was also observed for the mutant protein, F34Y/N37S, with two substitutions. These results indicated that the substitution of Asn37 with Ser can account, at least in part, for the characteristic time-courses of DEL. Moreover, replacement of Asn37 with Ser reduced the rate constant of transglycosylation. The substitution of the Asn37 residue may affect the transglycosylation activity of HEL.     

On the inhibition of hen egg-white lysozyme activity by aminoglycosidic antibiotics.

Lytic activity of hen egg-white lysozyme towards bacterial cells of Micrococcus lysodeikticus was pH-dependent inhibited by several aminoglycosidic antibiotics, the structure of which is related to the saccharidic substrates of the enzyme.Inhibition extent suggests the role of the positive charges of this type of antibiotics on the mechanism of lysozyme activity inhibition.     

Raman spectroscopic studies of hen egg-white lysozyme at high temperatures and pressures

In situ high-temperature, high-pressure Raman experiments on 3 mM (pH 5) aqueous solutions of hen egg-white (HEW) lysozyme show a decrease in the relative height of the 505 cm^–1 band associated with S-S stretching vibrations at 72°C (1 bar). The peak height changes are accompanied by significant band broadening, and the integrated band intensity does not change within experimental error. The effect of increased pressure at 72°C was to hinder broadening of the 505 cm^–1 band. HEW lysozyme (2.4 mM,pH 5) was also heated at 76°C, 80°C, and 95°C for different periods of time, and aliquots were quenched to room temperature for Raman and enzymatic activity measurements. After 9 hr at 76°C, the protein exhibits enzyme activity less than 50% of the initial value, and approximately 50% reduction in activity is achieved after 3 hr at 80°C or 1 hr at 95°C. The Raman results suggest that different irreversibly denatured conformations are attained during prolonged exposures at these different temperatures. It is apparent from these studies that the S-S stretch intensity is decreased irreversibly.     

Histidine-15 and lytic activity of lysozyme

The literature data on the activity of histidine-15 modified hen egg white lysozyme are conflicting: the modified enzyme is reported to have more activity, similar activity or less activity by different authors. Amino acid analysis had shown modification of the single His-15. Detailed activity studies on His-15-modified (by iodoacetic acid or diethyl pyrocarbonate) lysozyme have shown that the contradicting reports are due to the specific choices of ionic strengths and cell wall substrate concentrations and can be attributed to the substrate being negatively charged. Our analysis suggests that even though histidine-15 is far removed from the active site of lysozyme, its chemical modification or binding of the negatively-charged substrate near it, changes the conformation around the active site. However, the change in the optimum activity on chemically modifying His-15 is small.     

Exploring the inhibitory activity of short-chain phospholipids against amyloid fibrillogenesis of hen egg-white lysozyme

Amyloid fibrillogenesis is an important pathological feature of a group of degenerative human diseases. The 129-residue enzyme hen egg-white lysozyme has been shown to form fibrils in vitro at pH 2.0 and 55°C. In this research, using various spectroscopic techniques, light scattering, and transmission electron microscopy, we first examined the influence of short-chain phospholipids on the amyloid fibrillogenesis and the structural changes derived from hen lysozyme in vitro. Both model short-chain phospholipids were observed to mitigate the fibrillogenesis of hen lysozyme. Also, urea-induced unfolding results suggested that the susceptibility of hen lysozyme to conformational changes elicited by the denaturant was observed to decrease upon addition of short-chain phospholipids. Moreover, our molecular dynamics simulations results demonstrated that the observed inhibitory action of short-chain phosoholipids against hen lysozyme fibrillogenesis might be attributable to the interference of β-strand extension by the binding of phospholipids to lysozyme's β-sheet-rich region. We believe that the outcome from this study may contribute to a better understanding the molecular factors affecting amyloid fibrillogenesis and the molecular mechanism(s) of the interactions between phospholipids/lipids and amyloid-forming proteins.     

Pressure-dependent changes in the solution structure of hen egg-white lysozyme

The "rules" governing protein structure and stability are still poorly understood. Important clues have come from proteins that operate under extreme conditions, because these clarify the physical constraints on proteins. One obvious extreme is pressure, but so far little is known of the behavior of proteins under pressure, largely for technical reasons. We have therefore developed new methodology for calculating structure change in solution with pressure, using NMR chemical shift changes, and we report the change in structure of lysozyme on going from 30 bar to 2000 bar, this being the first solution structure of a globular protein under pressure. The alpha-helical domain is compressed by approximately 1%, due to tighter packing between helices. The interdomain region is also compressed. By contrast, the beta-sheet domain displays very little overall compression, but undergoes more structural distortion than the alpha-domain. The largest volume changes tend to occur close to hydrated cavities. Because isothermal compressibility is related to volume fluctuation, this suggests that buried water molecules play an important role in conformational fluctuation at normal pressures, and are implicated as the nucleation sites for structural changes leading to pressure denaturation or channel opening.     

Crystal structure of hen egg-white lysozyme at a hydrostatic pressure of 1000 atmospheres

The crystal structure of tetragonal hen egg-white lysozyme at a hydrostatic pressure of 1000 atmospheres has been determined by X-ray diffraction to a nominal resolution of 2 A. The crystals, originally grown in 0.83 M-NaCl, had to be transferred to 1.4 M-NaCl to prevent crystal cracking at 300 to 400 atm. The a and b axes of the unit cell contracted by 0.6%, whilst the c axis increased by 0.1%. The unit cell volume contracted by 1.1%. Both the 1 atm and the 1000 atm structures were refined by restrained least-squares to yield final R factors of 14.9% in each case. Since the data were collected by an accurate difference protocol, the change in structure is considered to be more accurate than the absolute structure. The probable accuracy of the atomic shifts is shown to be +/- 0.06 A. The estimated volume decrease of the whole molecule corresponded to an isothermal compressibility of 4.7 X 10(-3) kbar-1. The contraction was non-uniformly distributed. Domain 2 (residues 40 to 88) was essentially incompressible, whilst domain 1 (residues 1 to 39, 89 to 129) had a compressibility of 5.7 X 10(-3) kbar-1. The interdomain region was also compressible. The average B factor decreased about 1 A2 at 1000 atm, but there was a wide range of decreases and increases in individual values. The pressure-induced deformation was analyzed with difference distance matrices. The beta-sheet (residues 42 to 60) and helix 2 (residues 24 to 36) were deformed the least under pressure. The other helices were more deformed and one loop region (residues 61 to 87) actually appeared to expand. The main-chain atoms of the beta-sheet and helix 2 were used to perform a least-squares superposition of the 1 atm and 1000 atm models. The root-mean-square pressure-induced shift for all atoms was 0.2 A, with a few atoms moving more than 1 A. There was no evidence for co-ordinated movement about the hinge axis defined by alpha carbon atoms 38 and 97. The 1 atm and 1000 atm refined structures included 151 and 163 ordered water molecules, respectively. The changes in these ordered water molecules and the mean compressibility of all of the solvent in the crystal will be described elsewhere.     

Process evaluations and economic analyses of recombinant human lysozyme and hen egg-white lysozyme purifications

Human lysozyme and hen egg-white lysozyme have antibacterial, antiviral, and antifungal properties with numerous potential commercial applications. Currently, hen egg-white lysozyme dominates low cost applications but the recent high-level expression of human lysozyme in rice could provide an economical source of lysozyme. This work compares human lysozyme and hen egg-white lysozyme adsorption to the cation exchange resin, SP-Sepharose FF, and the effect of rice extract components on lysozyme purification. With one exception, the dynamic binding capacities of human lysozyme were lower than those of hen egg-white at pH 4.5, 6, and 7.5 with ionic strengths ranging from 0 to 100 mM (5-20 mS). Ionic strength and pH had a similar effect on the adsorption capacities, but human lysozyme was more sensitive to these two factors than hen egg-white lysozyme. In the presence of rice extract, the dynamic binding capacities of human and hen egg-white lysozymes were reduced by 20-30% and by 32-39% at pH 6. Hen egg-white lysozyme was used as a benchmark to compare the effectiveness of human lysozyme purification from transgenic rice extract. Process simulation and cost analyses for human lysozyme purification from rice and hen egg-white lysozyme purification from egg-white resulted in similar unit production costs at 1 ton per year scale.     

Effects of dithiothreitol on the amyloid fibrillogenesis of hen egg-white lysozyme

At least 25 human proteins can fold abnormally to form pathological deposits that are associated with several degenerative diseases. Despite extensive investigation on amyloid fibrillation, the detailed molecular mechanisms remain rather elusive and there are currently no effective cures for treating these amyloid diseases. The present study examined the effects of dithiothreitol on the fibrillation of hen egg-white lysozyme (HEWL). Our results revealed that the fibrillation of hen lysozyme was significantly inhibited by reduced dithiothreitol (DTT^red) while oxidized dithiothreitol (DTT^ox) had no anti-aggregating activity. Effective inhibitory activity against hen lysozyme fibrillation was observed only when DTT^red was added within 8 days of incubation. Our results showed that the initial addition of DTT^red interacted with HEWL, leading to a loss in conformational stability. It was concluded from our findings that DTT^red-induced attenuation of HEWL fibrillation may be associated with disulfide disruption and extensive structural unfolding of HEWL. Our data may contribute to rational design of effective therapeutic strategies for amyloid diseases.     

Effect of curcumin on the amyloid fibrillogenesis of hen egg-white lysozyme

At least twenty human proteins can fold abnormally to form pathological deposits that are associated with several degenerative diseases. Despite extensive investigation on amyloid fibrillogenesis, its detailed molecular mechanisms remain unknown. This study is aimed at exploring the inhibitory activity of curcumin against the fibrillation of hen lysozyme. We found that the formation of amyloid fibrils at pH 2.0 in vitro was inhibited by curcumin in a dose-dependent manner. Moreover, quenching analysis confirmed the existence of an interaction between curcumin and lysozyme, and Van't Hoff analysis indicated that the curcumin–lysozyme interaction is predominantly governed by Van Der Waals force or hydrogen bonding. Curcumin was also found to acquire disaggregating ability on preformed lysozyme fibrils. Finally, we observed that curcumin pre-incubated at 25 °C for at least 7 days inhibited lysozyme fibrillogenesis better than untreated curcumin and the enhanced inhibition against HEWL fibrillation might be attributed to the presence of dimeric species.     

An X-ray study of the physiological-temperature form of hen egg-white lysozyme at 2 A resolution

The structure of the high-temperature orthorhombic form of hen egg-white lysozyme has been determined at 2.0 A resolution. Initial images of the molecule were obtained at 6.0 A resolution both by double isomorphous replacement and by molecular replacement with use of the known structure of the room-temperature tetragonal lysozyme. The initial model thus obtained (R = 0.52 at 6.0 A) was refined first as a rigid body at 6.0 A and then by restrained least squares at 2.5 A and later at 2.0 A resolution. The final model (R = 0.23 at 2.0 A) was compared with that of the tetragonal form: the structures are very similar with a root mean square difference in superimposed alpha-carbon coordinates of 0.46 A. There are, however, differences which are caused by a crystal contact involving the upper part of this active site in the high-temperature orthorhombic form. Because of this, residues Trp 62 and Pro 70 are much better ordered than in the tetragonal form, where they are exposed to solvent. These differences can partly explain the difficulty of inhibitor-binding in high-temperature orthorhombic crystals, but do not seem to reflect the particular behaviour of lysozyme in solution at high temperature.     

Equilibrium and kinetic folding of hen egg-white lysozyme under acidic conditions

The equilibrium and kinetic folding of hen egg-white lysozyme was studied by means of circular dichroism spectra in the far- and near-ultraviolet (UV) regions at 25 degrees C under the acidic pH conditions. In equilibrium condition at pH 2.2, hen lysozyme shows a single cooperative transition in the GdnCl-induced unfolding experiment. However, in the GdnCl-induced unfolding process at lower pH 0.9, a distinct intermediate state with molten globule characteristics was observed. The time-dependent unfolding and refolding of the protein were induced by concentration jumps of the denaturant and measured by using stopped-flow circular dichroism at pH 2.2. Immediately after the dilution of denaturant, the kinetics of refolding shows evidence of a major unresolved far-UV CD change during the dead time (<10 ms) of the stopped-flow experiment (burst phase). The observed refolding and unfolding curves were both fitted well to a single-exponential function, and the rate constants obtained in the far- and near-UV regions coincided with each other. The dependence on denaturant concentration of amplitudes of burst phase and both rate constants was modeled quantitatively by a sequential three-state mechanism, U<-->I<-->N, in which the burst-phase intermediate (I) in rapid equilibrium with the unfolded state (U) precedes the rate-determining formation of the native state (N). The role of folding intermediate state of hen lysozyme was discussed.