The amino acid sequence of satyr tragopan lysozyme and its activity

The amino acid sequence of satyr tragopan lysozyme and its activity was analyzed. Carboxymethylated lysozyme was digested with trypsin and the resulting peptides were sequenced. The established amino acid sequence had three amino acid substitutions at positions 103 (Asn to Ser), 106 (Ser to Asn), and 121 (His to Gln) comparing with Temminck's tragopan lysozyme and five amino acid substitutions at positions 3 (Phe to Tyr), 15 (His to Leu), 41 (Gln to His), 101 (Asp to Gly) and 103 (Asn to Ser) with chicken lysozyme. The time course analysis using N-acetylglucosamine pentamer as a substrate showed a decrease of binding free energy change, 1.1 kcal/mol at subsite A and 0.2 kcal/mol at subsite B, between satyr tragopan and chicken lysozymes. This was assumed to be responsible for the amino acid substitutions at subsite A-B at position 101 (Asp to Gly), however another substitution at position 103 (Asn to Ser) considered not to affect the change of the substrate binding affinity by the observation of identical time course of satyr tragopan lysozyme with turkey and Temminck's tragopan lysozymes that carried the identical amino acids with chicken lysozyme at this position. These results indicate that the observed decrease of binding free energy change at subsites A-B of satyr tragopan lysozyme was responsible for the amino acid substitution at position 101 (Asp to Gly).     

Multiple role of hydrophobicity of tryptophan-108 in chicken lysozyme: structural stability, saccharide binding ability, and abnormal pKa of glutamic acid-35.

Trp108 of chicken lysozyme is in van der Waals contact with Glu35, one of two catalytic carboxyl groups. The role of Trp108 in lysozyme function and stability was investigated by using mutant lysozymes secreted from yeast. By the replacement of Trp108 with less hydrophobic residues, Tyr (W108Y lysozyme) and Gln (W108Q lysozyme), the activity, saccharide binding ability, stability, and pKa of Glu35 were all decreased with a decrease in the hydrophobicity of residue 108. Namely, at pH 5.5 and 40 degrees C, the activities of W108Y and W108Q lysozymes against glycol chitin were 17.3 and 1.6% of that of wild-type lysozyme, and their dissociation constants for the binding of a trimer of N-acetyl-D-glucosamine were 7.4 and 309 times larger than that of wild-type lysozyme, respectively. For the reversible unfolding at pH 3.5 and 30 degrees C, W108Y and W108Q lysozymes were less stable than wild-type lysozyme by 1.4 and 3.6 kcal/mol, respectively. As for the pKa of Glu35, the values for W108Y and W108Q lysozymes were found to be lower than that for wild-type lysozyme by 0.2 and by 0.6 pKa unit, respectively. The pKa of Glu35 in lysozyme was also decreased from 6.1 to 5.4 by the presence of 1-3 M guanidine hydrochloride, or to 5.5 by the substitution of Asn for Asp52, another catalytic carboxyl group. Thus, both the hydrophobicity of Trp108 and the electrostatic interaction with Asp52 are equally responsible for the abnormally high pKa (6.1) of Glu35, compared with that (4.4) of a normal glutamic acid residue.(ABSTRACT TRUNCATED AT 250 WORDS)     

The role of binding subsite A in reactions catalyzed by hen egg-white lysozyme

The role of binding subsite A, located at the terminal of the six binding subsites of hen egg-white lysozyme, in substrate binding and catalytic reactions was investigated by kinetic studies using a chemical modification method. Computer simulation showed that, although subsite A participates in the binding of the substrate, a decrease in the affinity of subsite A to the sugar residue does not cause a lowering of the rate of substrate consumption but changes the mode of the reaction by changing the distribution of the products formed. The binding free energies of subsites for Asp-101-modified lysozymes were estimated by data-fitting from the experimental time-courses. The contribution of Asp-101 in hen egg-white lysozyme to the substrate binding at subsite A was estimated to correspond to a binding free energy of about -3 kJ/mol, 30% of the total binding free energy of subsite A. Modification of Asp-101 affected not only the binding free energy of subsite A but also that of subsite C.     

Redesign of the substrate-binding site of hen egg white lysozyme based on the molecular evolution of C-type lysozymes.

On the basis of the molecular evolution of hen egg white, human, and turkey lysozymes, three replacements (Trp62 with Tyr, Asn37 with Gly, and Asp101 with Gly) were introduced into the active-site cleft of hen egg white lysozyme by site-directed mutagenesis. The replacement of Trp62 with Tyr led to enhanced bacteriolytic activity at pH 6.2 and a lower binding constant for chitotriose. The fluorescence spectral properties of this mutant hen egg white lysozyme were found to be similar to those of human lysozyme, which contains Tyr at position 62. The replacement of Asn37 with Gly had little effect on the enzymatic activity and binding constant for chitotriose. However, the combination of Asn37----Gly (N37G) replacement with Asp101----Gly (D101G) and Trp62----Tyr (W62Y) conversions enhanced bacteriolytic activity much more than each single mutation and restored hydrolytic activity toward glycol chitin. Consequently, the mutant lysozyme containing triple replacements (N37G, W62Y, and D101G) showed about 3-fold higher bacteriolytic activity than the wild-type hen lysozyme at pH 6.2, which is close to the optimum pH of the wild-type enzyme.     

Role of the loop structure of the catalytic domain in rice class I chitinase

In the three-dimensional structure of a rice class I chitinase (OsChia1b) determined recently, a loop structure (loop II) is located at the end of the substrate-binding cleft, and is thus suggested to be involved in substrate binding. In order to test this assumption, deletion of the loop II region from the catalytic domain of OsChia1b and replacement of Trp159 in loop II with Ala were carried out. The loop II deletion and the W159A mutation increased hydrolytic activity not only towards (GlcNAc)6 but also towards polysaccharide substrates. Similar results were obtained for kcat/Km values determined for substrate reduced-(GlcNAc)5. The two mutations shifted the splitting positions in (GlcNAc)6 to the reducing end side, but the shift was less intensive in the Trp mutant. Theoretical analysis of the reaction time course indicated that sugar residue affinity at the +3 subsite was reduced from -2 kcal/mol to +0.5 kcal/mol by loop II deletion. Reduced affinity at the +3 subsite might enhance the release of product fragments, resulting in higher turnover and higher enzymatic activities. Thus, we concluded that loop II is involved in sugar residue binding at the +3 subsite, but that Trp159 itself appears to contribute only partly to sugar residue interaction at the subsite.     

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.     

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.     

Effect of chemical modifications of tryptophan residues on the folding of reduced hen egg-white lysozyme

The effects of chemical modifications of Trp62 and Trp108 on the folding of hen egg-white lysozyme from the reduced form were investigated by means of the sulfhydryl-disulfide interchange reaction at pH 8 and 40 degrees C. The folding of reduced lysozyme was monitored by following the recovery of the original activity. Under the conditions employed, the apparent first-order rate constant for the folding of reduced lysozyme was not changed by the modifications of both Trp62 and Trp108 and the folding was completed within 30 min. However, the extent of the correct folding was changed by the modification of Trp62 but not by that of Trp108. Native and oxindolealanine108 lysozymes recovered 80 and 81% of their original activities after 30-min refolding, respectively, but Trp62-modified lysozymes recovered their activities to a lesser extent than native and oxindolealanine108 lysozymes. The recovered activities of Trp62-modified lysozymes after 30-min refolding were 63% for oxindolealanine62 lysozyme, 65% for delta 1-carboxamidomethylthiotryptophan62 lysozyme, and 52% for delta 1-carboxymethylthiotryptophan62 lysozyme. These results suggest that Trp62 is important for preventing the misfolding of reduced lysozyme, but that neither Trp62 nor Trp108 is involved in the rate-determining step (the slowest step) in the folding pathway. A decrease in the hydrophobic nature of Trp62 seems to increase the misfolding and thus to decrease the extent of the correct folding of reduced lysozyme. A mechanism for the involvement of Trp62 in the folding pathway of reduced lysozyme is proposed.     

1H-NMR study of the intramolecular interaction of a substrate analogue covalently attached to aspartic acid-101 in lysozyme

We prepared the lysozyme derivative in which the beta-carboxyl group of Asp101 was modified with alpha-O-methyl N-glycylglucosaminide as an amide by means of the carbodimide reaction (alpha-MGG lysozyme). Since Asp101 residue is located at the edge of the active site cleft, a 1H-NMR study was carried out for this derivative in order to investigate the interaction between the introduced substituent and the active site cleft. It was confirmed that the alpha-MGG moiety sat in the active site cleft in alpha-MGG lysozyme from the reduction of line broadening of the NH-proton of Trp63 located in the active site cleft, the remarkable chemical shift change of the methyl group of the alpha-MGG moiety upon adding a trimer of N-acetyl-D-glucosamine [(NAG)3], and the NOE between the C6-proton resonance of Trp63 and the methyl resonance of the alpha-MGG moiety. Furthermore, alpha-MGG lysozyme had increased thermal stability compared with native lysozyme. Therefore, it was concluded that the alpha-MGG moiety covalently attached to Asp101 interacted with the active site cleft to increase the thermal stability of lysozyme.     

Preparation and properties of a lysozyme derivative in which two domains are cross-linked intramolecularly between Trp62 and Asp101.

A lysozyme derivative in which two domains were cross-linked intramolecularly was newly prepared by means of a two-step reaction. First, the beta-carboxyl group of Asp101 in lysozyme was selectively modified with 2-(2-pyridyldithio)ethylamine in the presence of 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide hydrochloride. After reduction of the pyridyldithio moiety of Asp101 modified lysozyme at pH 4.5 with dithiothreitol, the derivative was allowed to cross-link intramolecularly by reaction with 1,3-dichloroacetone at pH 7. Intramolecularly cross-linked lysozyme thus formed was purified by gel chromatography followed by ion-exchange chromatography. Based on the results of 1H-NMR and peptide analyses, it was concluded that Asp101 was cross-linked to Trp62 with a -CH2COCH2SCH2CH2NH-bridge in this derivative. The derivative showed minor but distinct activity against Micrococcus lysodeikticus and glycol chitin. Its melting temperature for thermal denaturation was higher by 7.3 degrees than that of native lysozyme at pH 3.     

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.     

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.     

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.     

Structure and binding properties of hen lysozyme modified at tryptophan 62

The structure of a derivative of hen egg-white lysozyme (EC 3.2.1.17) modified by N-bromosuccinimide at Trp62 has been studied by both ¹H nuclear magnetic resonance spectroscopy and X-ray crystallography. It was shown that this modification, changing the tryptophan residue to an oxindolealanine² residue, only causes minor structural changes at the site of the modification, and that the overall structure of the native enzyme is maintained in the derivative. Both diastereomers of the oxindolealanine-62 lysozyme were observed by the two methods employed, in accordance with previous observations (Norton & Allerhand, 1976). The pK values of the catalytically important carboxyl groups of Glu35 and Asp52 were identical in the native enzyme and its derivative. However, the modified enzyme is virtually inactive in the hydrolysis of the cell-wall mucopolysaccharide of Micrococcus lysodeikticus. The binding of N-acetylglucosamine oligosaccharides to both native lysozyme and Ox-62 lysozyme was studied by nuclear magnetic resonance spectroscopy, observing the perturbations on the lysozyme ¹H n.m.r. resonances, and differences in the perturbations of the two systems demonstrated that binding of (GlcNAc)₃ in particular was not identical in the two systems. The structure of Ox-62 lysozyme-(GlcNAc)₃ was studied by X-ray crystallography and it was shown that only two GlcNAc residues make contact with the enzyme, binding the reducing end residue in a similar mode as the α-anomeric form of GlcNAc binds to the native enzyme (Blake et al., 1967a). On the basis of the results obtained by X-ray crystallography and ¹H n.m.r. spectroscopy, the lack of enzymatic activity of the Ox-62 lysozyme arises from the obstruction by the oxindolealanine residue of sub-site B of the active site, preventing productive binding of the substrate.     

Self-association of lysozyme. Thermochemical measurements: effect of chemical modification of Trp-62, Trp-108, and Glu-35.

Heats of dilution and of saccharide binding for hen egg white lysozyme have been measured at 30 degrees, 0.1 ionic strength, and pH 7 over the range 3 to 95 mg of protein/ml. The concentration dependence of the apparent relative molar enthalpy of lysozyme derived from these results gives the thermodynamic parameters for the formation of an intermolecular contact in an indefinite (head-to-tail) self-association process as delta G 0 = -3.9 kcal/mol, delta H 0 = -6.4 kcal/mol, and delta S 0 = -8,3 e.u. Oxindolealanine-62-lysozyme does not undergo self-association reactions that can be detected calorimetrically. This derivative reacts with native lysozyme to form hybrid polymeric species with free energy and enthalpy of interaction similar to those for the polymers of native lysozyme. These results are consistent with the intermolecular contact in the self-assocaition of lysozyme being asymmetric (head-to-tail). The heat of dilution of the derivative of lysozyme in which Glu-35 is blocked as the ester with oxindolealanine-108 is like that observed for native lysozyme in acid solution and is independent of pH. The concentration difference spectrum that develops through self-association is of the shape expected for introduction of an indole chromophore into a charge-free region of the intermolecular contact. The foregoing results indicate that Glu-35 and Trp-62 are part of the contact, that perturbation of Trp-108 does not make a principle contribution to the concentration difference spectrum, and that no acid group other than Glu-35 is perturbed by self-association. There is a small change in the heat of (GlcNAc)3 binding over the range 0.005 to 0.034 M saccharide. These data give the value of -1 kcal/mol for the enthalpy change for formation of the 2:1 saccharide-enzyme complex (ES2) from ES and S.     

Enhanced bacteriolytic activity of hen egg-white lysozyme due to conversion of Trp62 to other aromatic amino acid residues

Tryptophan at the 62nd position (Trp62) of hen egg-white lysozyme is an amino acid residue whose action is essential for its enzymatic activity. Its indole ring may possibly come into direct contact with sugar residues of the substrate, and thus contribute significantly to substrate binding. For further elucidation of its role in catalytic processes, this amino acid was converted to other aromatic residues, such as Tyr, Phe, and His, by site-directed mutagenesis. All the mutations were found to enhance the bacteriolytic activity but to decrease the hydrolytic activity toward an artificial substrate, glycol chitin. Such a change in substrate preference appears remarkable considering the smaller size of the aromatic residue on the mutant enzyme at the 62nd position.     

Binding of N-acetyl-chitotriose to human lysozyme.

The interaction of N-acetyl-chitotriose ((GlcNAc)3) with human lysozyme [EC 3.2.1.17] was studied at various pH values by measuring changes in the circular dichroic (CD) band at 294 or 255 nm and the data were compared with the results for hen and turkey lysozymes reported previously (Kuramitsu et al. (1974) J. Biochem.76, 671-683; Kuramitsu et al. (1975) J. Biochem. 77, 291-301). The pH dependence of the binding constant of (GlcNAc)3 to human lysozyme was different from those for hen and turkey lysozymes. The catalytic carboxyls of human lysozyme, Asp 52 and Glu 35, were not perturbed on binding of (GlcNAc)3. This is consistent with the previous findings that the macroscopic pK values of Asp 52 and Glu 35 of human lysozyme are 3.4 and 6.8 at 0.1 ionic strength and 25 degrees and were unchanged on complexing with (GlcNAc)3. An ionizable group with pK 4.5, which participates in the binding of (GlcNAc)3 to hen lysozyme and was assigned as Asp 101, did not participate in the binding of the saccharide to human lysozyme. Between pH 9 and 11, the binding constants of (GlcNAc)3 to hen lysozyme remained unchanged, whereas perturbation of an ionizable group with pK 10.5 to 10.0 was observed for human lysozyme. This group may be Tyr 62 in the active-site cleft. The binding constants of (GlcNAc)3 to human lysozyme molecules having different microscopic protonation forms, with respect to the catalytic carboxyls, were estimated using the binding constants obtained in the present experiments and the microscopic ionization constants of the catalytic carboxyls obtained previously. All four species of human lysozyme had similar binding constants to (GlcNAc)3. This result is different from those for hen and turkey lysozymes.     

Turkey egg white lysozyme. Free energy, enthalpy, and steady state kinetics of reaction with N-acetylglucosamine oligosaccharides.

Equilibrium and calorimetric studies of substrate binding to turkey egg white (TEW) lysozyme were carried out at 30degrees as a function of pH (2 to 9) and ligand size (monosaccharide to hexasaccharide of N-acetylglucosamine). Steady state kinetic measurements using the N-acetylglucosamine hexasaccharide were carried out as a function of pH (2 to 9) and temperature (20-60degrees). These experiments allow comparison of the properties of TEW lysozyme with those of the hen egg white (HEW) enzyme reported previously (Banerjee, S. K., Holler, E., Hess, G. P., and Rupley, J. A. (1975) J. Biol. Chem. 250, 4355-4367, and references therein). The free energies and enthalpies of oligosaccharide binding are the same for TEW and HEW lysozymes at pH 2 but are less negative for TEW lysozyme at pH 5. The pH dependence of the binding of (GlcNAc)3 and higher oligomers to TEW lysozyme is like that for the binding of beta-methyl-N-acetylglucosaminide to TEW lysozyme. These data indicate that oligosaccharide ligands bind identically with HEW and TEW lysozymes, except for the interactions of residue 101, which is aspartic acid in the HEW protein and glycine in the TEW protein (Larue, J. N., and Speck, J. C., Jr. (1970) J. Biol. Chem. 245, 1985-1991). The pH dependence of kcat is described by apparent pK values of 3.9 and 6.8 and a maximum value of kcat of 0.135 s-1. A value of 21.0 kcal/mol was calculated for deltaH from the temperature dependence of kcat. These values and the dependence of the transglycosylation reaction on acceptor concentration are within experimental error the same as those for HEW lysozyme. The more acid pK seen in the pH rate profile reflects the ionization of Asp-52 in the lysozyme-(GlcNAc)6 complex. The pK of Asp-52 in the free protein is 0.3 pK unit lower. The essential identity of the active sites of the HEW and TEW enzymes, except for the Asp-101 interactions, allows estimation of the thermodynamic properties associated with formation of the two hydrogen bonds between Asp-101 and substrate as deltaG0 = -1.2 kcal/mol, DeltaH0 = -3.6 kcal/mol, and deltaS0 = -7.9 e.u.     

Structures of partridge egg-white lysozyme with and without tri-N-acetylchitotriose inhibitor at 1.9 A resolution.

The three-dimensional structures of native partridge egg-white lysozyme (PEWL) and PEWL complexed with tri-N-acetylchitotriose inhibitor have been determined crystallographically and refined at 1.9 A resolution. Crystals of native and complexed protein are isomorphous and have space group and cell dimensions that are identical to those of hen egg-white lysozyme (HEWL) under similar crystallization conditions. Full occupancy of the trisaccharide in the inhibitor complex has allowed definitive modeling and refinement of all three sugar residues, located at subsites A, B, and C in the PEWL active site. A comparison has been made with HEWL/inhibitor complexes in which coordinates were either not refined (Blake CCF, et al., 1967, Proc R Soc B 167:378-388) or were refined at partial occupancy (Cheetham JC, Artymiuk PJ, Phillips DC, 1992, J Mol Biol 224:613-628). Although the loop comprising residues 70-75 is located on the surface of the protein and not near the active site, it appears to be affected indirectly by trisaccharide binding such that the loop shifts toward the active site and becomes relatively immobilized. The source of this loop movement appears to be the anchoring of Trp62, located in the active site cleft, as it forms a hydrogen bond with O6 of the N-acetylglucosamine at site C. Good electron density for the trisaccharide in the PEWL complex structure shows that Asp 101 is involved in hydrogen bonding interactions with the terminal sugar residue.     

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.