Enzymatic synthesis of p-nitrophenyl N,N',N',N',N'-pentaacetyl-beta-chitopentaoside in water-methanol system; significance as a substrate for lysozyme assay

Transchitooligosylation from (GlcNAc)5 to the 4-position of PNP-GlcNAc was efficiently induced through lysozyme catalysis in an aqueous solution containing methanol with a high concentration. Use of the aqueous methanol system in this reaction not only guaranteed solubility of PNP-GlcNAc substrate, but also resulted in a remarkable increase in PNP-(GlcNAc)5 production. PNP-(GlcNAc)5 was substrate for lysozyme assay compared with PNP-(GlcNAc)4.     

Enzymatic properties of rhea lysozyme

Rhea lysozyme was analyzed for its enzymatic properties both lytic and oligomer activities to reveal the structural and functional relationships of goose type lysozyme. Rhea lysozyme had the highest lytic activity at pH 6, followed by ostrich and goose at pH 5.5-6, whereas the optimum of cassowary was at pH 5. pH profile was correlated to the net charge of each molecule surface. On the other hand, the pH optimum for oligomer substrate was found to be pH 4, indicating the mechanism of rhea catalysis as a general acid. The time-course of the reaction was studied using beta-1,4-linked oligosaccharide of N-acetylglucosamine (GlcNAc) with a polymerization degree of n ((GlcNAc)n) (n=4, 5, and 6) as the substrate. This enzyme hydrolyzed (GlcNAc)6 in an endo-splitting manner, which produced (GlcNAc)3+(GlcNAc)3 predominating over that to (GlcNAc)2+ (GlcNAc)4. This indicates that the lysozyme hydrolyzed preferentially the third glycosidic linkage from the nonreducing end. Theoretical analysis has shown the highest rate constant value at 1.5 s-1 with (GlcNAc)6. This confirmed six substrate binding subsites as goose lysozyme (Honda, Y., and Fukamizo, T., Biochim. Biophys. Acta, 1388, 53-65 (1998)). The different binding free energy values for subsites B, C, F, and G from goose lysozyme might responsible for the amino acid substitutions, Asn122Ser and Phe123Met, located at the subsite B.     

p-nitrophenyl penta-N-acetyl-beta-chitopentaoside as a novel synthetic substrate for the colorimetric assay of lysozyme

p-Nitrophenyl beta-glycosides of N-acetylchitooligosaccharides (PNP-(GlcNAc)n n = 3-5) were examined as substrates for lysozyme [EC 3.2.1.17]. The enzyme released predominantly p-nitrophenyl N-acetyl-beta-D-glucosaminide (PNP-GlcNAc) from each substrate. Furthermore, the initial rate of PNP-GlcNAc formation in lysozyme-catalyzed hydrolysis of p-nitrophenyl penta-N-acetyl-beta-chitopentaoside (PNP-(GlcNAc)5) was about 350 and 25 times faster than those of p-nitrophenyl tri-N-acetyl-beta-chitotrioside (PNP-(GlcNAc)3) and p-nitrophenyl tetra-N-acetyl-beta-chitotetraoside (PNP-(GlcNAc)4), respectively. From these results, a new colorimetric assay method of lysozyme using PNP-(GlcNAc)5 as a substrate was developed on the basis of the determination of p-nitrophenol liberated from the substrate by lysozyme through a coupled reaction involving beta-N-acetylhexosaminidase (NAHase). The assay system gave a linear dose-response curve in the range of 2-120 micrograms of lysozyme in a 15-60 min incubation. The present assay was not significantly influenced by the ionic strength of the medium and was reproducible. This method using PNP-(GlcNAc)5 as a substrate was shown to be useful for lysozyme assay.     

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.     

Participation of the catalytic carboxyls, Asp 52 and Glu 35, and Asp 101 in the binding of substrate analogues to hen lysozyme.

The interactions of the substrate analogues, GlcNAc, beta-methyl GlcNAc, (GlcNAc)2, and (GlcNAc)3, with turkey egg-white lysozyme [ED 3.2.1.17], in which the Asp 101 of hen lysozyme is replaced by Gly, were studied at various pH values by measuring changes in the circular dichroic (CD) band at 295 nm. Results were compared with those for hen egg-white lysozyme. The modes of binding of these substrate analogues to turkey lysozyme were very similar to those hen lysozyme except for the participation of Asp 101 in hen lysozyme. The ionization constants of the catalytic carboxyls, Glu 35 and Asp 52, in the turkey lysozyme-(GlcNAc)3 complex were determined by measuring the pH dependence of the CD band at 304 nm, which originates from Trp 108 near the catalytic carboxyls. The ionization behavior of the catalytic carboxyls of turkey lysozyme in the presence and absence of (GlcNAc)3 was essentially the same as that for hen lysozyme. The pH dependence of the binding constant of (GlcNAc)3 to hen lysozyme was compared with that to turkey lysozyme between pH 2 and 8. The pH dependence of the binding constant for (GlcNAc)3 to turkey lysozyme could be interpreted entirely in terms of perturbation of catalytic carboxyls. In the case of hen lysozyme, it was interpreted in terms of perturbation of the catalytic carboxyls and Asp 101 in the substrate-binding site. The pK values of Asp 101 in hen lysozyme and the hen lysozyme-(GLcNAc)3 complex were 4.5 and 3.4, respectively. The binding constants of (GlcNAc)3 to lysozyme molecules with different microscopic protonation forms, with respect to the catalytic carboxyls, were estimated. The binding constant of lysozyme, in which Asp 52 and Glu 35 are deprotonated, to (GlcNAc)3 was the smallest. The other three species had similar binding constant to (GlcNAc)3.     

Reaction of N-acetylglucosamine oligosaccharides with lysozyme. Temperature, pH, and solvent deuterium isotope effects; equilbrium, steady state, and pre-steady state measurements*.

Temperature jump and stopped flow methods were used to study at pH 7.0 the temperature dependence of elementary steps of the reactions of lysozyme with the beta(1 yields 4)-linked trimer, tetramer, and hexamer of N-acetylglucosamine. The steady state rate of cleavage of the hexasaccharide was determined as a function of temperature (5 degrees-40 degrees) and pH(2 to 8) in H-2O solution and as a function of pD(2.5 to 9.5) at 40 degrees in D-2O solution. The apparent enthalpies of the two ionizations of apparent pK 3.8 and 6.7 observed in measurements of k are 0 to 2 kcal/mol. The energy of activation determined for the pH optimum is 21.5 kcal/mol. The solvent deuterium isotope effect measured for k at the pH (pD) optimum is 1.5 And reflects isotope effects on pre-equilibrium steps and on the rate-determining step. Transfer from H-2O to D-2O solution produces 0.2 to 0.4 kcal/mol more negative free energies of saccharide binding and no changes in the enthalpies of binding. Pre-steady state, steady state, and equilibrium measurements indicate a pathway for the reaction of lysozyme with hexasaccharide. The results define for this mechanism the complete free energy profile and an essentially complete enthalpy profile. Three of the five observable ES complexes are present at nearly equal concentrations. The free energies of the transition states are within a range of 3 kcal. The enthalpies of productive enzyme-substrate complexes are about 5 kcal/mol greater than the enthalpies of nonproductive complexes. Changes in tryptophan fluorescence were observed for each elementary step, and changes in pK of Glu-35 for the isomerizations of nonproductive and productive complexes. The signal changes during formation of nonproductive complexes are the same for the oligosaccharides (ClcNAc)3 to (GlcNAc)6. The changes for productive complexes are similar but not identical with saccharides (GlcNAc)4 to (GlcNAc)6. Correlations of the present data with previous crystallographic and solution measurements indicate the structures of productive and nonproductive ES complexes and suggest that full interaction of the substrate with the enzyme active site is established in the rate-determining step.     

Expression of a synthetic gene coding for ostrich egg-white lysozyme in Pichia pastoris and its enzymatic activity

To investigate the structure-function relationships of goose-type lysozyme, a gene coding for ostrich egg-white lysozyme (OEL) was designed based on the published amino acid sequence and constructed by assembling 32 chemically synthesized oligonucleotides. To obtain the recombinant OEL (rOEL), the synthetic gene was fused to the alpha-factor signal peptide in the expression vector pPIC9K and expressed in the methylotrophic yeast Pichia pastoris. The secreted protein from the transformed yeast was found to be processed at three different sites, including the correct site. The correctly processed rOEL was purified to homogeneity and shown to be indistinguishable from the authentic form in terms of circular dichroism (CD) spectrum and enzyme activity. Furthermore, the time-course of the reaction catalyzed by OEL was studied using (GlcNAc)(n) (n = 5 and 6) as the substrate and compared to that of goose egg-white lysozyme (GEL) [Honda and Fukamizo (1998) BIOCHIM: Biophys. Acta 1388, 53-65]. OEL hydrolyzed (GlcNAc)(6) in an endo-splitting manner producing mainly (GlcNAc)(2), (GlcNAc)(3), and (GlcNAc)(4), and cleavage to (GlcNAc)(3) + (GlcNAc)(3) predominated over that to (GlcNAc)(2) + (GlcNAc)(4). This indicates that OEL hydrolyzes preferentially the third glycosidic linkage from the nonreducing end of (GlcNAc)(6) as in the case of GEL. The cleavage pattern seen for (GlcNAc)(5) was similar to that seen for (GlcNAc)(6). Theoretical analysis of the reaction time-course for OEL revealed that the binding free energy values for subsites B, E, and G were different between OEL and GEL, although these lysozymes were estimated to have the same type of subsite structure.     

Enzymatic synthesis of an α-chitin-like substance via lysozyme-mediated transglycosylation

The enzymatic synthesis of an α-chitin-like substance via a non-biosynthetic pathway has been achieved by transglycosylation in an aqueous system of the corresponding substrate, tri-N-acetylchitotriose [(GlcNAc)(3)] for lysozyme. A significant amount of water-insoluble product precipitated out from the reaction system. MALDI-TOFMS analysis showed that the resulting precipitate had a degree of polymerization (DP) of up to 15 from (GlcNAc)(3). Solid-state (13)C NMR analysis revealed that the resulting water-insoluble product is a chitin-like substance consisting of N-acetylglucosamine (GlcNAc) residues joined exclusively in a β-(1→4)-linked chain with stringent regio-/stereoselection. X-ray diffraction (XRD) measurement as well as (13)C NMR analysis showed that the crystal structure of synthetic product corresponds to α-chitin with a high degree of crystallinity. We propose that the multiple oligomers form an α-chitin-like substance as a result of self-assembly via oligomer-oligomer interaction when they precipitate.     

Amino acid sequence of Egyptian goose egg-white lysozyme and effects of amino acid substitution on the enzymatic activity

The amino acid sequence of Egyptian goose lysozyme (EGL) from egg-white and its enzymatic properties were analyzed. The established sequence had the highest similarity to wood duck lysozyme (WDL) with five amino acid substitutions, and had eighteen substitutions difference from hen egg-white lysozyme (HEL). Tyr34 and Gly37 were found at subsites E and F of the active site when compared with HEL. The experimental time-course characteristics of EGL against the N-acetylglucosamine pentamer substrate, (GlcNAc)(5), revealed higher production of (GlcNAc)(4) and lower production of (GlcNAc)(2) when compared with HEL. The saccharide-binding ability of subsites A-C in EGL was also found to be weaker than in HEL. An analysis of the enzymatic reactions of five mutants in respect of positions 34, 37 and 71 in HEL indicated the time-course characteristics of EGL to be caused by the combination of three substitutions (F34Y, N37G and G71R) between HEL and EGL. A computer simulation of the EGL-catalyzed reaction suggested that the time-course characteristics of EGL resulted from the difference in the binding free energy for subsites A, B, E and F and the rate constant of transglycosylation between EGL and HEL.     

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.     

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.     

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

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

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.     

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.     

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.     

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.     

Low-molecular-weight substrate for the lysozyme of T4 bacteriophage.

It has been shown that muropeptide CB, the chemically defined product of Escherichia coli B murein digestion by phage lambda endolysin, is the substrate for T4 lysozyme. This is the tetrasaccharide GlcNAc-MurNAc-GlcNAc-anMurNAc in which the carboxyl groups of MurNAc and anMurNAc residues are substituted by tetrapeptide LAla-DGlu-msA2pm-DAla (MurNAc = N-acetylmuramic acid, GlcNAc = N-acetyl-D-glucosamine, anMurNAc = 1,6-anhydro-N-acetylmuramic acid, LAla = L-alanine, DGlu = D-glutamic acid, msA2pm = meso-diaminopimelic acid). The substrate contains one bond hydrolysable by T4 lysozyme. The products of hydrolysis are the easily identifiable disaccharide muropeptides C6 (GlcNAc-MurNAc-LAla-DGlu-msA2pm-DAla) and CA (GlcNAc-anMurNac-LAla-DGlu-msA2pm-DAla). Thus the substrate may be used for the specific identification of murein N-acetylmuramoylhydrolases of the T4 lysozyme type, as well as for any quantitative measurement of the enzymatic reaction.     

Catalytic reaction mechanism of goose egg-white lysozyme by molecular modelling of enzyme-substrate complex

Despite the low similarity between their amino acid sequences, the core structures of the fold between chicken-type and goose-type lysozymes are conserved. However, their enzymatic activities are quite different. Both of them exhibit hydrolytic activities, but the goose-type lysozyme does not exhibit transglycosylation activity. The chicken-type lysozyme has a retaining-type reaction mechanism, while the reaction mechanism of the goose-type lysozyme has not been clarified. To clarify the latter mechanism, goose egg-white lysozyme (GEL)-N-acetyl-D-glucosamine (GlcNAc)6 complexes were modelled and compared with hen egg-white lysozyme (HEL)-(GlcNAc)6 complexes. By systematic conformational search, 48 GEL-(GlcNAc)6 complexes were modelled. The right and left side, and the amino acid residues in subsites E-G were identified in GEL. The GlcNAc residue D could bind towards the right side without distortion and there was enough room for a water molecule to attack the C1 carbon of GlcNAc residue D from alpha-side in the right side and not for acceptor molecule. The result of molecular dynamics simulation suggests that GEL would be an inverting enzyme, and Asp97 would act as a second carboxylate and that the narrow space of the binding cleft at subsites E-G in GEL may prohibit the sugar chain to bind alternative site that might be essential for transglycosylation.     

Functional and structural effects of mutagenic replacement of Asn37 at subsite F on the lysozyme-catalyzed reaction

To investigate the functional role of subsites E and F in lysozyme catalysis, Asn37 of hen egg-white lysozyme (HEL), which is postulated to participate in sugar residue binding at the right-sided subsite F through hydrogen bonding, was replaced by Ser or Gly by site-directed mutagenesis. The mutations of Asn37 neither significantly affected the binding constant for chitotriose nor the enzymatic activity toward the substrate glycol chitin. However, kinetic analysis with the substrate N-acetylglucosamine pentamer, (GlcNAc)(5), revealed that the conversion of Asn37 to Gly decreased the binding free energies for subsites E and F, while the conversion to Ser increased the substrate affinity at subsite F. It was further found that the rate constant of transglycosylation was reduced by these mutations. These results suggest that Asn37 is involved not only in substrate binding at subsite F but also in transglycosylation activity. No remarkable change in the tertiary structure except the side chain of the 37th residue was detected on X-ray analysis of the mutant proteins, indicating that the alterations in the enzymatic function between the wild type and mutant enzymes depend on limited structural change around the substitution site. It is thus speculated that the slight conformational difference in the side chain of position 37 may affect the substrate and acceptor binding at subsites E and F, leading to lower the efficiency of the transglycosylation activities of the mutant proteins.     

A structural model for (GlcNAc)2 translocation via a periplasmic chitooligosaccharide-binding protein from marine Vibrio bacteria

VhCBP is a periplasmic chitooligosaccharide-binding protein mainly responsible for translocation of the chitooligosaccharide (GlcNAc)₂ across the double membranes of marine bacteria. However, structural and thermodynamic understanding of the sugar-binding/-release processes of VhCBP is relatively less. VhCBP displayed the greatest affinity toward (GlcNAc)₂, with lower affinity for longer-chain chitooligosaccharides [(GlcNAc)_3–4]. (GlcNAc)₄ partially occupied the closed sugar-binding groove, with two reducing-end GlcNAc units extending beyond the sugar-binding groove and barely characterized by weak electron density. Mutation of three conserved residues (Trp³⁶³, Asp³⁶⁵, and Trp⁵¹³) to Ala resulted in drastic decreases in the binding affinity toward the preferred substrate (GlcNAc)₂, indicating their significant contributions to sugar binding. The structure of the W513A–(GlcNAc)₂ complex in a ‘half-open’ conformation unveiled the intermediary step of the (GlcNAc)₂ translocation from the soluble CBP in the periplasm to the inner membrane–transporting components. Isothermal calorimetry data suggested that VhCBP adopts the high-affinity conformation to bind (GlcNAc)₂, while its low-affinity conformation facilitated sugar release. Thus, chitooligosaccharide translocation, conferred by periplasmic VhCBP, is a crucial step in the chitin catabolic pathway, allowing Vibrio bacteria to thrive in oceans where chitin is their major source of nutrients.