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.     

Histidine-114 at subsites E and F can explain the characteristic enzymatic activity of guinea hen egg-white lysozyme

The courses of the reaction catalyzed by guinea hen egg-white lysozyme (GHL), in which Asn113 and Arg114 at subsites E and F in hen egg-white lysozyme (HEL) are replaced by Lys and His, respectively, was studied with the substrate N-acetylglucosamine pentamer, (GlcNAc)5. Although GHL was found to retain the main-chain folding similar to HEL as judged from CD spectroscopy, the courses of GHL showed increased production of (GlcNAc)4 and reduced production of (GlcNAc)2 when compared with HEL. To identify critical residue(s) involved in the alteration in the courses of GHL, two mutant enzymes as to subsites E and F in HEL, N113K and R114H, were prepared by site-directed mutagenesis. Kinetic analysis of these mutants revealed that the mutation of Asn113 to Lys had little effect on the courses of HEL, while the Arg114 to His mutation completely reproduced the courses of GHL, demonstrating that His114 in GHL is the key residue responsible for the characteristic courses of GHL. Computer simulation of the reaction courses of the R114H mutant revealed that this substitution decreased not only the binding free energies for subsites E and F, but also the rate constant of transglycosylation. The Arg residue at position 114 may play an important role in the transglycosylation activity of HEL.     

Binding of substrate analogs to hen egg-white lysozyme with an ester linkage between Glu 35 and Trp 108.

The interactions of the substrate analogs beta-methyl-GlcNAc, (GlcNAc)2, and (GlcNAc)3 with hen egg-white lysozyme [EC 3.2.1.17] in which an ester linkage had been formed between Glu 35 and Trp 108 (108 ester lysozyme), were studied by the circular dichroic and fluorescence techniques, and were compared with those for intact lysozyme. The binding constants of beta-methyl-GlcNAc and (GlcNAc)2 to 108 ester lysozyme were essentially the same as those for intact lysozyme in the pH range of 1 to 5. Above pH 5, the binding constants of these saccharides to 108 ester lysozyme did not change with pH, while the binding constants to intact lysozyme decreased. This indicates that Glu 35 (pK 6.0 in intact lysozyme) participates in the binding of these saccharides. The extent and direction of the pK shifts of Asp 52 (pK 3.5), Asp 48 (pK 4.4), and Asp 66 (pK 1.3) observed when beta-methyl-GlcNAc is bound to 108 ester lysozyme were the same as those for intact lysozyme. The participation of Asp 101 and Asp 66 in the binding of (GlcNAc)2 to 108 ester lysozyme was also the same as that for intact lysozyme. These findings indicate that the conformations of subsites B and C are not changed by the formation of the ester linkage. On the other hand, the binding constants of (GlcNAc)3 to 108 ester lysozyme were higher than those for intact lysozyme at all pH values studied. This result is interpreted in terms of an increase in the affinity for a GlcNAc residue of subsite D, which is situated near the esterified Glu 35.     

卵清溶菌酶淀粉样纤维形成的研究

淀粉样纤维与老年性痴呆症、帕金森病和非神经性组织淀粉样变性病等人类疾病相关。运用ThT荧光、刚果红结合、远紫外圆二色、透射电镜的方法研究了不同条件下卵清溶菌酶（HEWL）淀粉样纤维的形成。实验结果表明pH值2．0是HEWL淀粉样纤维形成的必要条件,HEWL淀粉样纤维的形成是一个典型的浓度依赖型过程。三氟乙醇（TFE）对HEWL淀粉样纤维形成影响结果表明中低浓度（低于40％）的TFE加速了溶菌酶淀粉样纤维的形成,其中5％～15％（v／v）的TFE促进效果最为显著,大大缩短了淀粉样纤维的成核期;而高浓度的TFE（50％）则完全抑制了溶菌酶淀粉样纤维的形成。透射电镜直接观察了溶菌酶淀粉样纤维的形态,不加TFE时溶菌酶淀粉样纤维聚集成簇,形成相互缠绕的成熟纤维,而10％的TFE存在时,观察到的形态则主要是短的原纤维,且没有发生纤维的相互交联实验。结果表明溶菌酶形成相互缠绕的成熟纤维主要由弱的疏水相互作用来驱动。     

Binding of divalent copper ions to aspartic acid residue 52 in hen egg-white lysozyme

Divalent copper was found to inhibit non-competitively the lysis of Micrococcus lysodeikticus cells by hen egg-white lysozyme, with an inhibition constant K_a= 3.8 × 10²m^?1. The association constants of Cu²⁺ for lysozyme and for a derivative of lysozyme in which tryptophan residue 108 was selectively modified, were measured spectrofluorimetrieally and found to be 1.8 × 10²m^?1 and 1.0 × 10³m^?1, respectively. The electron spin resonance spectrum of Cu²⁺ was not affected by the addition of lysozyme, whereas many new lines appeared on addition of the modified protein. This was interpreted as evidence for the binding of Cu²⁺ in the neighbourhood of tryptophan 108. To unequivocally establish the site of ligation of Cu²⁺, crystals of lysozyme soaked in Cu²⁺ were examined by X-ray crystallography and the results compared to those obtained from crystals of native lysozyme. Cu²⁺ was found to be located 2 to 3 Å from the carboxyl side-chain of aspartic acid 52, 5 Å from the carboxyl of glutamic acid 35 and about 7 Å from tryptophan 108.The addition of a saccharide inhibitor to lysozyme was found to increase the association constant of Cu²⁺ for lysozyme from a value of 1.8 × 10²m^?1 to 6.0 × 10²m^?1. This finding was interpreted as indicative of a change in conformation around tryptophan 108 and glutamic acid 35 induced by the interaction of saccharides with the enzyme, which affects the metal binding properties of aspartic acid 52.     

Ionic-strength-dependent substrate inhibition of the lysis of Micrococcus luteus by hen egg-white lysozyme

The kinetics of lysis of Micrococcus luteus by hen egg-white lysozyme in dilute buffer media is characterized by pronounced substrate inhibition. This effect occurs within the complete pH range where lysozyme activity is detectable. The electrostatic potential of the negatively charged cell-wall proteoglycan increases with decreasing ionic strength, resulting in an enhanced affinity between proteoglycan and lysozyme and probably favouring multipoint substrate attachment. For the lysozyme-catalyzed hydrolysis of cell-wall proteoglycan three plausible mechanisms of substrate inhibition can be postulated. Two out of the three models fit our experimental data, the simplest of the two providing the most rigorous information on the kinetic parameters Km, V and Ki. Three graphical methods consistent with the chosen model were applied for preliminary parameter estimation and the constants obtained were compared to those from nonlinear least-squares analysis. If substrate inhibition is neglected it is shown that serious bias is imposed upon the parameters.     

Hydrogen exchange in native and denatured states of hen egg-white lysozyme.

The hydrogen exchange kinetics of 68 individual amide protons in the native state of hen lysozyme have been measured at pH 7.5 and 30 degrees C by 2D NMR methods. These constitute the most protected subset of amides, with exchange half lives some 10(5)-10(7) times longer than anticipated from studies of small model peptides. The observed distribution of rates under these conditions can be rationalized to a large extent in terms of the hydrogen bonding of individual amides and their burial from bulk solvent. Exchange rates have also been measured in a reversibly denatured state of lysozyme; this was made possible under very mild conditions, pH 2.0 35 degrees C, by lowering the stability of the native state through selective cleavage of the Cys-6-Cys-127 disulfide cross-link (CM6-127 lysozyme). In this state the exchange rates for the majority of amides approach, within a factor of 5, the values anticipated from small model peptides. For a few amides, however, there is evidence for significant retardation (up to nearly 20-fold) relative to the predicted rates. The pattern of protection observed under these conditions does not reflect the behavior of the protein under strongly native conditions, suggesting that regions of native-like structure do not persist significantly in the denatured state of CM6-127 lysozyme. The pattern of exchange rates from the native protein at high temperature, pH 3.8 69 degrees C, resembles that of the acid-denatured state, suggesting that under these conditions the exchange kinetics are dominated by transient global unfolding. The rates of folding and unfolding under these conditions were determined independently by magnetization transfer NMR methods, enabling the intrinsic exchange rates from the denatured state to be deduced on the basis of this model, under conditions where the predominant equilibrium species is the native state. Again, in the case of most amides these rates showed only limited deviation from those predicted by a simple random coil model. This reinforces the view that these denatured states of lysozyme have little persistent residual order and contrasts with the behavior found for compact partially folded states of proteins, including an intermediate detected transiently during the refolding of hen lysozyme.     

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.     

Cooperative folding of the isolated alpha-helical domain of hen egg-white lysozyme.

Proteins in the alpha-lactalbumin and c-type lysozyme family have been studied extensively as model systems in protein folding. Early formation of the alpha-helical domain is observed in both alpha-lactalbumin and c-type lysozyme; however, the details of the kinetic folding pathways are significantly different. The major folding intermediate of hen egg-white lysozyme has a cooperatively formed tertiary structure, whereas the intermediate of alpha-lactalbumin exhibits the characteristics of a molten globule. In this study, we have designed and constructed an isolated alpha-helical domain of hen egg-white lysozyme, called Lyso-alpha, as a model of the lysozyme folding intermediate that is stable at equilibrium. Disulfide-exchange studies show that under native conditions, the cysteine residues in Lyso-alpha prefer to form the same set of disulfide bonds as in the alpha-helical domain of full-length lysozyme. Under denaturing conditions, formation of the nearest-neighbor disulfide bonds is strongly preferred. In contrast to the isolated alpha-helical domain of alpha-lactalbumin, Lyso-alpha with two native disulfide bonds exhibits a well-defined tertiary structure, as indicated by cooperative thermal unfolding and a well-dispersed NMR spectrum. Thus, the determinants for formation of the cooperative side-chain interactions are located mainly in the alpha-helical domain. Our studies suggest that the difference in kinetic folding pathways between alpha-lactalbumin and lysozyme can be explained by the difference in packing density between secondary structural elements and support the hypothesis that the structured regions in a protein folding intermediate may correspond to regions that can fold independently.     

Solvent isotope effects on the refolding kinetics of hen egg-white lysozyme.

Solvent isotope effects have been observed on the in vitro refolding kinetics of a protein, hen lysozyme. The rates of two distinct phases of refolding resolved by intrinsic fluorescence have been found to be altered, to differing extents, in D2O compared with H2O, and experiments have been conducted aimed at assessing the contributions to these effects from various possible sources. The rates were found to be essentially independent of whether backbone amide nitrogens were protiated or deuterated, indicating that making and breaking of their hydrogen bonding interactions is not associated with a substantial isotope effect. Neither were the rates significantly affected by adding moderate concentrations of sucrose or glycerol to the refolding buffer, suggesting that viscosity differences between H2O and D2O are also unlikely to explain the isotope effects. The data suggest that different factors, acting in opposing directions, may be dominant under different conditions. Thus, the isotope effect on the rate-determining step was found to be qualitatively reversed on going to low pH, suggesting that one component is probably associated with changes in the environments of carboxylate groups in forming the folding transition state. This term disappears at low pH as these groups are protonated and an opposing effect then dominates. It was not possible to identify this other effect on the basis of the present data, but a dependence of the hydrophobic interaction on solvent isotopic composition is a likely candidate.     

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.     

Effects of copolypeptides on amyloid fibrillation of hen egg-white lysozyme

The fibrillation of hen egg-white lysozyme (HEWL) in the absence and presence of simple, unstructured D,L-lysine-co-glycine (D,L-Lys-co-gly) and D,L-lysine-co-L-phenylalanine (D,L-Lys-co-Phe) copolypeptides was studied by using a variety of analytical techniques. The attenuating and decelerating effects on fibrillation are significantly dependent on the polypeptide concentration and the composition ratios in the polypeptide chain. Interestingly, D,L-Lys-co-gly and D,L-Lys-co-Phe copolypeptides with the same composition ratio have comparable attenuating effects on fibrillation. The copolypeptide with highest molar fraction of glycine residue exhibits the strongest suppression of HEWL fibrillation. The copolypeptide has the highest hydrophobic interacting capacity due to the more molar ratio of apolar monomer in the polymer backbone. The major driving forces for the association of HEWL and copolypeptides are likely to be hydrogen bonding and hydrophobic interactions, and these interactions reduce the concentration of free protein in solution available to proceed to fibrillation, leading to the increase of lag time and attenuation of fibrillation. The results of this work may contribute to the understanding of the molecular factors affecting amyloid fibrillation and the molecular mechanism(s) of the interactions between the unstructured polypeptides and the amyloid proteins.     

Structural and functional properties of hen egg-white lysozyme deamidated by protein engineering.

The structural and functional properties of lysozymes genetically deamidated at positions 103 (N103D) and 106 (N106D) were studied by a protein engineering technique. The wild-type and mutant lysozymes were expressed in Saccharomyces cerevisiae and purified from the cultivation medium in two steps by cation-exchange chromatography on CM-Toyopearl. The lytic activity of deamidated lysozymes was almost the same as that of wild lysozyme, although the optimal pH of activity was slightly shifted to lower pH by the deamidation. The Gibbs free energy changes of unfolding (delta G) at 20 degrees C for N103D and N106D were almost the same as that of wild-type. On the other hand, the structural flexibility of lysozymes, estimated by protease digestion, was significantly increased by the deamidation. The surface functional properties of deamidated lysozymes were considerably enhanced, compared to those of wild-type lysozyme. These results suggest that structural flexibility is an important governing factor in surface functional properties of proteins, regardless of their structural stability.     

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.