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.     

The identification by x-ray crystallography of the site of attachment of an affinity label to hen egg-white lysozyme

Tetragonal crystals of hen egg-white lysozyme were treated with the active sitedirected irreversible inhibitor 2′,3′epoxypropyl β-glycoside of N-acetyl-d-glucosamine, β(1→4)-linked dimer. The crystals were examined by X-ray crystallography, and the results compared to those obtained from crystals of the reversible complex formed between hen egg-white lysozyme and the β-phenyl glycoside of GlcNAc β(1→4)GlcNAc. It is concluded that the GlcNAc β(1→4)GlcNAc moiety of the irreversible inhibitor occupies subsites B and C in the active site of the enzyme, and that the inhibitor is linked covalently to the enzyme through the carboxyl side-chain of Asp 52.     

State of binding subsites in Asp 101-modified lysozymes

The environments of the binding subsites in Asp 101-modified lysozyme, in which glucosamine or ethanolamine is covalently bound to the carboxyl group of Asp 101, were investigated by chemical modification and nuclear magnetic resonance spectroscopy. Trp 62 in each of the native and the modified lysozymes was nitrophenylsulfenylated. The yield of the nitrophenylsulfenylated derivative from the lysozyme modified with glucosamine at Asp 101 (GlcN-lysozyme) was considerably lower than those from native lysozyme and from the lysozyme modified with ethanolamine at Asp 101 (EtN-lysozyme). These results suggest that Trp 62 in GlcN-lysozyme is less susceptible to nitrophenylsulfenylation. Kinetic analyses of the [Trp 62 and Asp 101]-doubly modified lysozymes indicated that the nitrophenylsulfenylation of Trp 62 in the native lysozyme, EtN-lysozyme, or GlcN-lysozyme decreased the sugar residue affinity at subsite C while increasing the binding free energy change by 2.7 kcal/mol, 1.5 kcal/mol, or 0.1 kcal/mol, respectively. Although the profile of tryptophan indole NH resonances in the 1H-NMR spectrum for EtN-lysozyme was not different from that for the native lysozyme, the indole NH resonance of Trp 62 in GlcN-lysozyme was apparently perturbed in comparison with that of native lysozyme. These results suggest that the environment of subsite C in GlcN-lysozyme is considerably different from those in native lysozyme and EtN-lysozyme. The glucosamine residue attached to Asp 101 may contact the sugar residue binding site of the lysozyme, affecting the environment of subsite C.     

Discoidin I from Dictyostelium discoideum and Interactions with Oligosaccharides: Specificity, Affinity, Crystal Structures, and Comparison with Discoidin II

Discoidin I (DiscI) and discoidin II (DiscII) are N-acetylgalactosamine (GalNAc)-binding proteins from Dictyostelium discoideum. They consist of two domains: an N-terminal discoidin domain and a C-terminal H-type lectin domain. They were cloned and expressed in high yield in recombinant form in Escherichia coli. Although both lectins bind galactose (Gal) and GalNAc, glycan array experiments performed on the recombinant proteins displayed strong differences in their specificity for oligosaccharides. DiscI and DiscII bind preferentially to Gal/GalNAcβ1-3Gal/GalNAc-containing and Gal/GalNAcβ1-4GlcNAcβ1-6Gal/GalNAc-containing glycans, respectively. The affinity of the interaction of DiscI with monosaccharides and disaccharides was evaluated using isothermal titration calorimetry experiments. The three-dimensional structures of native DiscI and its complexes with GalNAc, GalNAcβ1-3Gal, and Galβ1-3GalNAc were solved by X-ray crystallography. DiscI forms trimers with involvement of calcium at the monomer interface. The N-terminal discoidin domain presents a structural similarity to F-type lectins such as the eel agglutinin, where an amphiphilic binding pocket suggests possible carbohydrate-binding activity. In the C-terminal H-type lectin domain, the GalNAc residue establishes specific hydrogen bonds that explain the observed affinity (K_d = 3 × 10^− 4 M). The different specificities of DiscI and DiscII for oligosaccharides were rationalized from the different structures obtained by either X-ray crystallography or molecular modeling.     

A goose-type lysozyme from ostrich (Struthio camelus) egg white: multiple roles of His101 in its enzymatic reaction

A goose-type lysozyme from ostrich egg white (OEL) was produced by Escherichia coli expression system, and the role of His101 of OEL in the enzymatic reaction was investigated by NMR spectroscopy, thermal unfolding, and theoretical modeling of the enzymatic hydrolysis of hexa-N-acetylchitohexaose, (GlcNAc)₆. Although the binding of tri-N-acetylchitotriose, (GlcNAc)₃, to OEL perturbed several backbone resonances in the ¹H–¹⁵N HSQC spectrum, the chemical shift of the backbone resonance of His101 was not significantly affected. However, apparent pK_a values of His101 and Lys102 determined from the pH titration curves of the backbone chemical shifts were markedly shifted by (GlcNAc)₃ binding. Thermal unfolding experiments and modeling study of (GlcNAc)₆ hydrolysis using a His101-mutated OEL (H101A-OEL) revealed that the His101 mutation affected not only sugar residue affinities at subsites ?3 and ?2 but also the rate constant for bond cleavage. His101 appears to play multiple roles in the substrate binding and the catalytic reaction.     

A Novel Transition-state Analogue for Lysozyme, 4-O-β-Tri-N-acetylchitotriosyl Moranoline,Provided Evidence Supporting the Covalent Glycosyl-enzyme Intermediate

4-O-β-Di-N-acetylchitobiosyl moranoline (2) and 4-O-β-tri-N-acetylchitotriosyl moranoline (3) were produced by lysozyme-mediated transglycosylation from the substrates tetra-N-acetylchitotetraose, (GlcNAc)₄, and moranoline, and the binding modes of 2 and 3 to hen egg white lysozyme (HEWL) was examined by inhibition kinetics, isothermal titration calorimetry (ITC), and x-ray crystallography. Compounds 2 and 3 specifically bound to HEWL, acting as competitive inhibitors with K_i values of 2.01 × 10⁻⁵ and 1.84 × 10⁻⁶ m, respectively. From ITC analysis, the binding of 3 was found to be driven by favorable enthalpy change (ΔH_r°), which is similar to those obtained for 2 and (GlcNAc)₄. However, the entropy loss (−TΔS_r°) for the binding of 3 was smaller than those of 2 and (GlcNAc)₄. Thus the binding of 3 was found to be more favorable than those of the others. Judging from the K_d value of 3 (760 nm), the compound appears to have the highest affinity among the lysozyme inhibitors identified to date. X-ray crystal structure of HEWL in a complex with 3 showed that compound 3 binds to subsites −4 to −1 and the moranoline moiety adopts an undistorted ⁴C₁ chair conformation almost overlapping with the −1 sugar covalently bound to Asp-52 of HEWL (Vocadlo, Davies, G. J., Laine, R., and Withers, S. G. (2001) Nature 412, 835–838). From these results, we concluded that compound 3 serves as a transition-state analogue for lysozyme providing additional evidence supporting the covalent glycosyl-enzyme intermediate in the catalytic reaction.     

Crystallographic determination of the mode of binding of oligosaccharides to T4 bacteriophage lysozyme: Implications for the mechanism of catalysis

Phage lysozyme has catalytic activity similar to that of hen egg white lysozyme, but the amino acid sequences of the two enzymes are completely different.The binding to phage lysozyme of several saccharides including N-acetylglucosamine (GlcNAc), N-acetylmuramic acid (MurNAc) and (GlcNAc)₃ have been determined crystallographically and shown to occupy the pronounced active site cleft. GlcNAc binds at a single location analogous to the C site of hen egg white lysozyme. MurNAc binds at the same site. (GlcNAc)₃ clearly occupies sites B and C, but the binding in site A is ill-defined.Model building suggests that, with the enzyme in the conformation seen in the crystal structure, a saccharide in the normal chair configuration cannot be placed in site D without incurring unacceptable steric interference between sugar and protein. However, as with hen egg white lysozyme, the bad contacts can be avoided by assuming the saccharide to be in the sofa conformation. Also Asp20 in T4 lysozyme is located 3 Å from carbon C₍₁₎ of saccharide D, and is in a position to stabilize the developing positive charge on a carbonium ion intermediate. Prior genetic evidence had indicated that Asp20 is critically important for catalysis. This suggests that in phage lysozyme catalysis is promoted by a combination of steric and electronic effects, acting in concert, The enzyme shape favors the binding in site D of a saccharide with the geometry of the transition state, while Asp20 stabilizes the positive charge on the oxocarbonium ion of this intermediate. Tn phage lysozyme, the identity of the proton donor is uncertain. In contrast to hen egg white lysozyme, where Glu35 is 3 Å from the glycosidic DOE bond, and is in a non-polar environment, phage lysozyme has an ion pair, Glull … Arg145, 5 Å away from the glycosidic oxygen. Possibly Glull undergoes a conformational adjustment in the presence of bound substrate, and acts as the proton donor. Alternatively, the proton might come from a bound water molecule.     

Quantitative studies of the non-productive binding of lysozyme to partially N-acetylated chitosans: Binding of large ligands to a one-dimensional binary lattice studied by a modified McGhee and von Hippel model

This paper considers the non-productive (inhibitory) binding of chitosans to lysozyme from chicken egg white. Chitosans are linear, binary heteropolysaccharides consisting of 2-acetamido-2-deoxy-β-d-glucose (GlcNAc; A-unit) and 2-amino-2-deoxy-β-d-glucose (GlcN, D-unit). The active site cleft of lysozyme can bind six consecutive sugar residues in subsites named A–F, and specific binding of chitosan sequences to lysozyme occurs with A-units in subsite C. Chitosans with different fractions of A-units (F_A) induced nearly identical changes in the ¹H NMR spectrum of lysozyme upon binding, and the concentration of bound lysozyme could be determined. The data were analysed using a modified version of the McGhee and von Hippel model for binding of large ligands to one-dimensional homogeneous lattices. The average value of the dissociation constant for different sequences that may bind to lysozyme (K^ave_D) was estimated, as well as the number of chitosan units covered by lysozyme upon binding. K^ave_D decreased with increasing F_A-values at pH* 3 and 4.5, while the opposite was true at pH* 5.5. Contributions from different hexamer sequences to K^ave_D of the chitosans were considered, and the data revealed interesting features with respect to binding of lysozyme to partially N-acetylated chitosans. The relevance of the present data with respect to understanding lysozyme degradation kinetics of chitosans is discussed.     

Product-identification and substrate-specificity studies of the GDP-l-fucose: 2-acetamido-2-deoxy-β-d-glucoside (fuc→asn-linked GlcNAc) 6-α-l-fucosyltransferase in a golgi-rich fraction from porcine liver

Golgi-rich membranes from porcine liver have been shown to contain an enzyme that transfers l-fucose in α-(1→6) linkage from GDP-l-fucose to the asparagine-linked 2-acetamido-2-deoxy-d-glucose r residue of a glycopeptide derived from human α₁-acid glycoprotein. Product identification was performed by high-resolution, ¹H-n.m.r. spectroscopy at 360 MHz and by permethylation analysis. The enzyme has been named GDP-l-fucose: 2-acetamido-2-deoxy-β-d-glucoside (Fuc→Asn-linked GlcNAc) 6-α-l-fucosyltransferase, because the substrate requires a terminal β-(1→2)-linked GlcNAc residue on the α-Man (1→3) arm of the core. Glycopeptides with this residue were shown to be acceptors whether they contained 3 or 5 Man residues. Substrate-specificity studies have shown that diantennary glycopeptides with two terminal β-(1→2)-linked GlcNAc residues and glycopeptides with more than two terminal GlcNAc residues are also excellent acceptors for the fucosyltransferase. An examination of four pairs of glycopeptides differing only by the absence or presence of a bisecting GlcNAc residue in β-(1→4) linkage to the β-linked Man residue of the core showed that the bisecting GlcNAc prevented 6-α-l-fucosyltransferase action. These findings probably explain why the oligosaccharides with a high content of mannose and the hybrid oligosaccharides with a bisecting GlcNAc residue that have been isolated to date do not contain a core l-fucosyl residue.     

1H nuclear magnetic resonance studies of the interaction of urea with hen lysozyme. Origins of the conformational change induced in hen lysozyme by N-acetylglucosamine oligosaccharides.

The interaction between hen lysozyme and urea has been investigated using 1H nuclear magnetic resonance spectroscopy. Chemical shift changes for resonances of a number of residues in the vicinity of the active site of the protein have been observed in the presence of urea prior to denaturation. These shifts are similar to those induced in the hen lysozyme spectrum by the specific binding of N-acetylglucosamine (GlcNAc) in site C of the active site cleft, indicating that urea and GlcNAc induce a similar conformational change in the enzyme. This implies that the conformational changes experienced by the enzyme on the binding of GlcNAc oligosaccharides are the consequence of interactions, possibly hydrogen bonding, involving the N-acetyl group of the sugar residue bound in site C, rather than the result of contacts between the protein and the pyranose rings of the oligosaccharides. This suggests that hen lysozyme employs an induced fit type mechanism to discriminate for N-acetylated saccharides as substrates.     

Specific carbodiimide-binding mechanism for the selective modification of the aspartic acid-101 residue of lysozyme in the carbodiimide-amine reaction

A mechanism for the selective modification of Asp-101 in hen egg-white lysozyme with an amine nucleophile catalyzed by 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide hydrochloride (EDC) was investigated using ethanolamine as a nucleophile at pH 5.0 and room temperature. In the presence of N-acetyl-D-glucosamine (NAG) and its oligomers [(NAG)n, n = 2 and 3] under the conditions with which about 90% of lysozyme was calculated to form complexes, the formation of Asp-101 modified lysozyme decreased markedly but to different degrees, that is (NAG)3 was the most and NAG the least effective. When the lysozyme derivative, in which Trp-62 in the active site cleft was oxidized to oxindolealanine (Ox-62 lysozyme), was used in place of native lysozyme, the formation of Asp-101 modified derivative decreased to about half, which was similar to the decrease in the presence of (NAG)2. In the presence of 0.5 M NaCl, on the other hand, the formation of Asp-101 modified lysozyme was considerably enhanced. From these observations, it is concluded that EDC binds to the active site cleft of lysozyme to specifically activate Asp-101. The affinity of EDC to the active site of lysozyme is partly due to the hydrophobic interaction of EDC with the Trp-62 residue at sub-site B of lysozyme. EDC is an activating reagent for carboxyl groups unlike most active site-directed reagents which produce final products directly. Therefore, the active site-directed nature of EDC was very useful because it made it possible to selectively introduce various amines as needed at a particular carboxyl group of lysozyme.     

Site-specific 13C-labeling of Trp 62 in hen egg-white lysozyme: preparation and 13C-NMR titration of [delta 1-13C]Trp 62-lysozyme.

The indole C-2(delta 1) carbon of Trp 62 in hen egg-white lysozyme was selectively labeled with 13C through a series of reactions involving N'-formylkynurenine 62-lysozyme with K13CN, NaBH4-reduction, and acid-catalyzed dehydration. [delta 1-13C]Trp 62-lysozyme in which Trp 62 is labeled with 90% 13C has the same chemical and enzymatic properties as the native protein. The reverted lysozyme gave a single 13C-NMR signal at 125 ppm. pH-titration of the 13C signal indicated a transition at pH 3.9 for the free enzyme. In the presence of (GlcNAc)3, the resonance signals were shifted 0.5-1 ppm upfield, and the transitions in the titration curve were observed at pH 3.9 and 6.5. Asp 52 and Glu 35 were assigned to the groups with pKas of 3.9 and 6.5, respectively. In [2-13C]AHT 62-lysozyme, which has 3-(2-amino-3-hydroxy-3H-[2-13C]indol-3-yl)alanine (AHT) at position 62, AHT 62 behaved quite differently from Trp 62 on pH-titration of the 13C-label. These results suggest that a conformational change around Trp 62 is induced upon ionization of the catalytic residue and that the structural flexibility of the side chain of this aromatic residue in the substrate binding site is closely related to the function of lysozyme.     

Product-identification and substrate-specificity studies of the GDP-l-fucose: 2-acetamido-2-deoxy-β-d-glucoside (fuc→asn-linked GlcNAc) 6-α-l-fucosyltransferase in a golgi-rich fraction from porcine liver

Golgi-rich membranes from porcine liver have been shown to contain an enzyme that transfers l-fucose in α-(1→6) linkage from GDP-l-fucose to the asparagine-linked 2-acetamido-2-deoxy-d-glucose r residue of a glycopeptide derived from human α₁-acid glycoprotein. Product identification was performed by high-resolution, ¹H-n.m.r. spectroscopy at 360 MHz and by permethylation analysis. The enzyme has been named GDP-l-fucose: 2-acetamido-2-deoxy-β-d-glucoside (Fuc→Asn-linked GlcNAc) 6-α-l-fucosyltransferase, because the substrate requires a terminal β-(1→2)-linked GlcNAc residue on the α-Man (1→3) arm of the core. Glycopeptides with this residue were shown to be acceptors whether they contained 3 or 5 Man residues. Substrate-specificity studies have shown that diantennary glycopeptides with two terminal β-(1→2)-linked GlcNAc residues and glycopeptides with more than two terminal GlcNAc residues are also excellent acceptors for the fucosyltransferase. An examination of four pairs of glycopeptides differing only by the absence or presence of a bisecting GlcNAc residue in β-(1→4) linkage to the β-linked Man residue of the core showed that the bisecting GlcNAc prevented 6-α-l-fucosyltransferase action. These findings probably explain why the oligosaccharides with a high content of mannose and the hybrid oligosaccharides with a bisecting GlcNAc residue that have been isolated to date do not contain a core l-fucosyl residue.     

Exploring the Chemical Space around 8-Mercaptoguanine as a Route to New Inhibitors of the Folate Biosynthesis Enzyme HPPK

As the second essential enzyme of the folate biosynthetic pathway, the potential antimicrobial target, HPPK (6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase), catalyzes the Mg^2+-dependant transfer of pyrophosphate from the cofactor (ATP) to the substrate, 6-hydroxymethyl-7,8-dihydropterin. Recently, we showed that 8-mercaptoguanine (8-MG) bound at the substrate site (K_D ∼13 µM), inhibited the S. aureus enzyme (SaHPPK) (IC₅₀ ∼ 41 µM), and determined the structure of the SaHPPK/8-MG complex. Here we present the synthesis of a series of guanine derivatives, together with their HPPK binding affinities, as determined by SPR and ITC analysis. The binding mode of the most potent was investigated using 2D NMR spectroscopy and X-ray crystallography. The results indicate, firstly, that the SH group of 8-MG makes a significant contribution to the free energy of binding. Secondly, direct N ⁹ substitution, or tautomerization arising from N ⁷ substitution in some cases, leads to a dramatic reduction in affinity due to loss of a critical N ⁹-H···Val46 hydrogen bond, combined with the limited space available around the N ⁹ position. The water-filled pocket under the N ⁷ position is significantly more tolerant of substitution, with a hydroxyl ethyl 8-MG derivative attached to N ⁷ (compound 21a) exhibiting an affinity for the apo enzyme comparable to the parent compound (K_D ∼ 12 µM). In contrast to 8-MG, however, 21a displays competitive binding with the ATP cofactor, as judged by NMR and SPR analysis. The 1.85 Å X-ray structure of the SaHPPK/21a complex confirms that extension from the N ⁷ position towards the Mg²⁺-binding site, which affords the only tractable route out from the pterin-binding pocket. Promising strategies for the creation of more potent binders might therefore include the introduction of groups capable of interacting with the Mg²⁺ centres or Mg²⁺ -binding residues, as well as the development of bitopic inhibitors featuring 8-MG linked to a moiety targeting the ATP cofactor binding site.     

1H-NMR study on the chitotrisaccharide binding to hen egg white lysozyme.

Interaction between hen egg white lysozyme and chitotrisaccharide was investigated by 1H-NMR spectroscopy using partially acetylated chitotrisaccharides and chemically modified lysozyme. Monoacetyl (GlcN-GlcN-GlcNAc), diacetyl (GlcN-GlcNAc-GlcNAc), or triacetyl chitotrisaccharide [(GlcNAc)3] was added to the lysozyme solution, and the changes in the 1H-NMR signals of the lysozyme were analyzed. Although many of the resonances were affected by addition of the saccharide, the most remarkable effect was seen on the signal of Trp28 C5H which is in a hydrophobic box adjacent to the saccharide-binding site. The signal shifted upfield by 0.2 ppm upon (GlcNAc)3 binding, whereas the chemical shift change of the signal resulting from binding of GlcN-GlcNAc-GlcNAc or GlcN-GlcN-GlcNAc was smaller than that resulting from (GlcNAc)3 binding. When the Asp101-modified lysozyme was used instead of the native lysozyme, the chemical shift change of the Trp28 C5H signal resulting from (GlcNAc)3 binding was also smaller than that for the native lysozyme. The chemical shift change of the signal reflects the conformational change of the hydrophobic box region which should synchronize with the movement of the binding site resulting from the saccharide binding. Therefore, the conformational change resulting from the saccharide binding might be reduced when the sugar residues located at binding subsites A and B of the lysozyme are deacetylated, as well as when Asp101 interacting with the sugar residues at the same subsites is modified.     

Effect of gem 2,2′-disubstitution and base in the formation of spiro- and ansa-1,3-propandioxy derivatives of cyclotriphosphazenes

The gem-dialkyl effect has been investigated in the reactions of cyclotriphosphazene, N₃P₃Cl₆1, with various 2,2′-derivatives of 1,3-propandiol, CXY(CH₂OH)₂, in either THF or DCM to form spiro (6-membered) and ansa (8-membered ring) derivatives. The reactions were made with a number of symmetrically-substituted (X = Y, methyl, ethyl, n-butyl and a malonate ester) and unsymmetrically-substituted (X ≠ Y, methyl/H, phenyl/H, methyl/n-propyl, ethyl/n-butyl and Br/NO₂) 1,3-propandiols. The products were analysed by ¹H and ³¹P NMR spectroscopy and some of the spiro and ansa derivatives were also characterized by X-ray crystallography. Reactions of 1 with unsymmetrically-substituted 1,3-propandiols results in the formation of two structural isomers of ansa-substituted compounds, both isomers (endo and exo) have been structurally-characterized by X-ray crystallography for the ethyl/n-butyl derivative. It is found that the regioselectivity of the reaction is changed when the base is changed. The relative proportions of spiro and ansa compounds formed under different reaction conditions were quantified by ³¹P NMR measurements of the reaction mixtures. The results were rationalised mainly in terms of the electronic effect of the substituents, whereas the steric effect has a secondary role in the formation of both spiro and ansa compounds.     

Sulfation of agarose from subantarctic Ahnfeltia plicata (Ahnfeltiales,Rhodophyta): studies of its antioxidant and anticoagulant properties in vitro and its copolymerization with acrylamide

Aqueous extraction of Ahnfeltia plicata collected in the Magellan ecoregion afforded agarose devoid of sulfate groups. This neutral agarose was subjected to sulfation with SO₃-pyridine complex, giving an aqueous soluble derivative with 35.5 % sulfate groups. Analysis by Fourier transform infrared spectroscopy (FT-IR) and by ¹H and ¹³C NMR spectroscopy indicated that this derivative was sulfated at positions C-6 of the β-galactopyranosyl residue and C-2 of the α-3,6-anhydrogalactopyranosyl residue and partially sulfated at position C-2 of the β residue. The antioxidant capacity of sulfated agarose was evaluated by the oxygen radical absorbance capacity (ORAC) method, ABTS radical cation, hydroxyl radicals, and chelating assays. This capacity of sulfated agarose toward peroxyl radicals was higher than that of commercial λ-carrageenan, while native agarose presented good activity, with an ORAC value similar to that of commercial κ-carrageenan. Sulfated agarose presented good antioxidant capacity toward other radicals. Copolymerization of sulfated agarose with acrylamide was achieved using ceric ammonium nitrate as initiator. NMR spectroscopy indicated grafting of polyacrylamide at position C-4 of β-galactopyranosyl residues.     

The role of the tryptophan-62 residue in the structure and function of lysozyme]

The thermostability and thermodinamics of formation of the enzyme-substrate complex of two oxidation products of chicken egg lysozyme with the tryptophane-62 residue modified to N'-formylkinurenine (with 2.5% activity) and kinurenine (with 27.5% activity) have been studied. In thermostability and pH effect on the substrate binding the lysozyme oxidation products do not differ from native lysozyme. The data obtained and thermodynamical characteristics of the enzyme-substrate complex formation suggest that the chemical nature of the 62 residue does not significantly affect the conformational properties of lysozyme, however, having a strongly pronounced effect on the binding of substrate and hence the total enzyme activity.     

Fluorescence polarization studies of lysozyme and lysozyme-saccharide complexes

The fluorescence polarization properties of hen egg white lysozyme and of an iodine oxidized derivative of lysozyme in which tryptophan-108 was selectively modified, were investigated. Using the addition law of anisotropy of mixed systems, the contribution of tryptophan-108 to the anisotropy spectrum of lysozyme and lysozyme-chitotetraose complex was separated. The rate of fluorescence polarization was studied as a function of pH. The major contribution to this rate is shown to arise from internal rotations of the indole side-chain of tryptophan-108 as well as from structural changes around tryptophan-62 and 63. From the dependence of the fluorescence polarization of lysozyme and IL with saccharide concentration, the existence of the simultaneous binding of two saccharide molecules to the enzyme cleft was inferred. At low chitotetraose concentration, the subsites A, B and C are occupied with an association constant of 8 × 10⁴m^?1 whereas at high saccharide concentration, both subsites A–B–C and E–F are occupied. The association constants of a series of saccharides to subsites E–F were measured and all found to be around 2 × 10²m^?1. The dependence of the rate of depolarization with saccharide concentration was determined and showed that, upon binding of the first saccharide molecule to subsites A, B and C, the rate of internal rotation of tryptophan-108 and tryptophan-62 and 63 was much reduced whereas upon further binding of a saccharide molecule in subsites E–F the rates are enhanced. This behaviour was interpreted as an indication that the binding of saccharide in subsites E–F induces changes in conformation of the enzyme which affect the entire active site architecture.