Stopped-flow fluorescence studies on saccharide binding to lysozyme.

The binding of the beta-1-4-linked trimer of N-acetyl-D-glucosamine to hen egg-white lysozyme was studied by rapid-reaction-kinetic methods with tryptophyl fluorescence observation of the transients. It was found that discrete segments of the fluorescence-difference spectrum from this reaction were perturbed at different time-points during the binding process. The results were interpretated as the formation of the initial complex, the fast phase of the reaction, perturbing the environment of tryptophan-62 and a subsequent and slower rearrangement of the initial complex perturbing the environment of tryptophan-108. At pH 4.4, the release of protons from aspartate-101 occurred during the rearrangement step of the binding reaction. A model for the reaction is presented (E, enzyme; L, ligand): (see article) The association of this ligand with lysozyme may be visualized in three-dimensional terms as initial complex-formation across the top of the active-site cleft followed by a diving motion of the ligand into the cleft.     

Differential scanning calorimetric studies on binding of N-acetyl-D-glucosamine to lysozyme

Differential scanning calorimetric (DSC) measurements were performed on the thermal denaturation of lysozyme and lysozyme complexed with N-acetyl-D-glucosamine (GlcNAc) at pH 5.00 (acetate buffer), 4.25 and 2.25 (Gly-HCl buffer). DSC data have been analyzed to obtain denaturation temperature T(d), enthalpy of denaturation DeltaH(D), heat capacity of denaturation DeltaC(pd) and cooperativity index eta. From these thermodynamic parameters, the binding constant K(L) and enthalpy of binding DeltaH(L), for the weak binding of lysozyme with GlcNAc have been determined. The values of K(L) and DeltaH(L) at pH 5.00 and 298 K are 42 +/- 4 M(-1) and -24 +/- 4 kJ mol(-1), respectively, and agree very well with the experimentally determined values from equilibrium and other studies. The binding constant has also been estimated by simulating the DSC curve with varying values of K(L) (T(d)) until it matches the experimental curve.     

Fluorescence polarization studies of saccharide binding to Wheat germ agglutinin and lysozyme

N-acetyl-D-glucosamine causes only slight increases in the polarization of Wheat germ agglutinin fluorescence at saturating levels whereas the disaccharide and trisaccharide produce increases in the polarization value from 0.116 to 0.151 and 0.154 respectively. These increases suggest that rotational motions of the tryptophan residue at the binding sites are being restricted by an interaction between these tryptophans and the bound sugars. A model of the nature and location of these interactions is discussed.Comparable results are obtained with lysozyme, which shows a larger effect upon binding of N-acetyl-D-glucosamine, but a maximal increase in polarization upon binding the corresponding disaccharide or trisaccharide.     

Dielectric behavior of lysozyme and ferricytochrome-c in water/ethylene-glycol solutions

This work deals with a dielectric study at radio frequencies of the influence at room temperature of two organic molecules, known as cryo-protectants, ethylene-glycol and glycerol, on conformational and dynamic properties of two model proteins, lysozyme (lys) from chicken egg-white and ferricytochrome-c (cyt-c) from horse heart. Cyt-c is a compact globular protein whereas lys is composed of two structural domains, separated by the active site cleft. Measurements were carried out at the fixed temperature of 20 degrees C varying the concentration of the cosolvent up to 90% w/w. From the analysis of the dielectric relaxation of the protein solution, the effective hydrodynamic radius and the electric dipole moment of the protein were calculated as a function of the cosolvent concentration. The data show that glycerol does not modify significantly the conformation of both proteins and cyt-c is also stable in the presence of ethylene-glycol. On the contrary ethylene-glycol strongly affects the dielectric response of lysozyme denoting a specific effect on its conformation and dynamics. The data are coherently interpreted hypothesizing that glycol molecule wedges between and separates the two domains of lys making them rotationally independent.     

Light-induced binding of riboflavin to lysozyme

Summary The photodynamic inactivation of lysozyme in air saturated H₂O and D₂O (phosphate buffer 0.05 M, pH 7.0) in the presence of methylene blue and riboflavin has been studied. When H₂O was replaced by D₂O a great increase in the rate of photoinactivation of lysozyme was observed. This finding, together with the fact that photooxidation is inhibited by singlet oxygen quenchers like NaN₃, suggests that these reactions occur via a singlet oxygen mechanism.During the course of the studies of the riboflavin sensitized photoinactivation of lysozyme, it was found that riboflavin is strongly bound to the enzyme as a result of illumination. This finding would explain the higher quantum yield observed when riboflavin is used, although this dye is bleached during irradiation.     

Volumetric characterization of tri-N-acetylglucosamine binding to lysozyme

Volumetric characteristics of protein recognition events determine the direction of pressure-induced shifts in the recognition reaction, while also providing insights into the structural, dynamic, and hydration changes. We report changes in volume, ΔV, and adiabatic compressibility, ΔK(S), accompanying the binding of tri-N-acetylglucosamine [(GlcNAc)(3)] to lysozyme at 25 °C in a pH 5.5 sodium acetate buffer. We interpret our measured changes in volume and compressibility in terms of changes in hydration and dynamic properties of the protein. On the basis of our ΔV data, we find that 79 ± 44 water molecules are released to the bulk from the hydration shells of the protein and the ligand. Our ΔK(S) data suggest a 4 ± 2% decrease in the mean-square fluctuations of the intrinsic volume of the protein, <δV(M)(2)> (or a 2% decrease in δV(M)). Thus, the trisaccharide-bound state of the enzyme is less hydrated, more rigid, and less dynamic compared to the unbound state. In general, we discuss the importance of volumetric insights into the molecular origins of protein recognition events.     

Permeability of lysozyme tetragonal crystals to water

Diffusion of water within cross-linked tetragonal crystals of hen egg-white lysozyme has been measured and simulated on a computer using the X-ray structure of water-filled channels within the crystal lattice. Relative to the self-diffusion coefficient of bulk water molecules, the experimental diffusion coefficient of water within the crystal was found to be 13 times reduced in the (001) crystallographic plane and 5 times reduced in the [001] direction. Comparison of the experimental and computer simulated diffusion coefficients shows that steric limitations for water diffusion are mostly responsible for this reduction of the water diffusion in the crystal, with the self-diffusion coefficient of intracrystalline water reduced by no more than 30–40% as compared to that of bulk water.     

Thermal-stimulated pressure and current studies of bound water in lysozyme.

The state of bound water in crystalized lysozyme was studied by four techniques: electret thermal depolarization currents, thermal-stimulated pressure, isothermal polization decay, and thermogravimetry. Hydration levels ranged from 0 to 40 mg water/g protein. Desorption of bound water dipoles was found to be the main process responsible for electrical depolarization. Two different binding sites for water were identified with long relaxation times at room temperature (order 10(2)s) and activation energies of 0.34 plus or minus 0.02 eV and 0.55 plus or minus 0.04 eV.     

Left-sided substrate binding of lysozyme: evidence for the involvement of asparagine-46 in the initial binding of substrate to chicken lysozyme.

The "right-sided" and "left-sided" substrate binding modes at the lower saccharide binding subsites (D-F sites) of chicken lysozyme were investigated by utilizing mutant lysozymes secreted from yeast. We constructed the following mutant lysozymes; "left-sided" substitution of Asn46 to Asp, deletion of Thr47, and insertion of Gly between Thr47 and Asp48 and "right-sided" substitution of Asn37 to Gly. Analyses of their activities and substrate binding abilities showed that Asn46 and Thr47 are involved in the initial enzyme-substrate complex and Asn37 is involved in the transition state. These results support an earlier proposal that interactions between substrate and residues at the left side of lysozyme stabilize a catalytically inactive enzyme-substrate complex, while interactions between substrate and residues at the right side stabilize the catalytically active complex [Pincus, M. R., & Scheraga, H. A. (1979) Macromolecules 12, 633-644]. These results are also consistent with the proposed kinetic mechanism for lysozyme reaction that the rearrangement of an initial enzyme-substrate complex (beta-complex) to another complex (gamma-complex) is required for catalytic hydrolysis [Banerjee S. K., Holler, E., Hess, G. P., & Rupley, J. A. (1975) J. Biol. Chem. 250, 4355-4367].     

Dielectric studies on water in solutions of purified lecithin vesicles

The complex permittivity of sonicated aqueous solutions of purified dimyristoylphosphatidylcholine has been measured as a function of frequency between 3 kHz and 40 GHz. The dielectric spectrum of the samples shows two dispersion/absorption regions, one centered at about 80 MHz the other at about 20.GHz (30°C). Otherwise than in previous studies no additional dispersion/absorption process has been found at frequencies below 10 MHz.The complex dielectric spectrum of the samples is discussed with respect to the dynamical state of solvent water in solutions of single-bilayer vesicles. The main relaxation time of the solvent water, τ₁ ((2πτ₁)^?1 ≈ 20 GHz), is smaller than that of pure water, τ_W, at the same temperature. This effect results from the action of internal depolarizing fields which obviously overcompensate and enhancement of τ₁ due to specific solute/solvent interactions (hydration) as had been previously found with micellar solutions of lysolecithins.It cannot be excluded, that some solvent water shows unusual dynamical behaviour. If there exists a substantial amount of such motionally perturbed water, however, it must be characterized by a relaxation time close to that of the phosphorylcholine zwitterions, τ₂ ((2πτ₂)^?1 ≈ 80 MHz).     

Theoretical studies of enzymic reactions: Dielectric,electrostatic and steric stabilization of the carbonium ion in the reaction of lysozyme

A general method for detailed study of enzymic reactions is presented. The method considers the complete enzyme-substrate complex together with the surrounding solvent and evaluates all the different quantum mechanical and classical energy factors that can affect the reaction pathway. These factors include the quantum mechanical energies associated with bond cleavage and charge redistribution of the substrate and the classical energies of steric and electrostatic interactions between the substrate and the enzyme. The electrostatic polarization of the enzyme atoms and the orientation of the dipoles of the surrounding water molecules is simulated by a microscopic dielectric model. The solvation energy resulting from this polarization is considerable and must be included in any realistic calculation of chemical reactions involving anything more than an isolated molecule in vacuo. Without it, acidic groups can never become ionized and the charge distribution on the substrate will not be reasonable. The same dielectric model can also be used to study the reaction of the substrate in solution. In this way the reaction in solution can be compared with the enzymic reaction.In this paper we study the stability of the carbonium ion intermediate formed in the cleavage of a glycosidic bond by lysozyme. It is found that electrostatic stabilization is an important factor in increasing the rate of the reaction step that leads to the formation of the carbonium ion intermediate. Steric factors, such as the strain of the substrate on binding to lysozyme, do not seem to contribute significantly.     

The stepwise binding of small molecules to proteins. Nuclear magnetic resonance and temperature jump studies of the binding of 4-(N-acetylaminoglucosyl)-N-acetylglucosamine to lysozyme.

The binding of 4-(N-acetylaminoglucosyl)-N-acetylglucosamine to lysozyme was studied by both nuclear magnetic resonance (NMR) and temperature-jump methods under comparable conditions. The NMR measurements on the inhibitor spectrum were carried out over a range of inhibitor concentrations including levels at which most of the inhibitor was bound to the enzyme. Data in this region were obtained by a novel difference method in conjunction with correlation spectroscopy. The results from the combination of both experimental techniques demonstrated the existence of a two-step binding mechanism and produced both values for all of the individual rate constants and also the NMR spectral data for the inhibitor in the two enzyme-inhibitor complexes. The later data characterize the environment experienced by the inhibitor at each stage in the binding process and thus provides both a three-dimensional and a dynamic picture of the interaction.