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.     

Amino Acid Sequence and Activity of Green Turtle (Chelonia mydas) Lysozyme

Green turtle lysozyme purified from egg white was sequenced and analyzed its activity. Lysozyme was reduced and pyridylethylated or carboxymethylated to digest with trypsin, chymotrypsin and V8 protease. The peptides yielded were purified by RP-HPLC and sequenced. Every trypsin peptide was overlapped by chymotrypsin peptides and V8 protease peptides. This lysozyme is composed of 130 amino acids including an insertion of a Gly residue between 47 and 48 residues when compared with chicken lysozyme. The amino acid substitutions were found at subsites E and F. Namely Phe34, Arg45, Thr47, and Arg114 were replaced by Tyr, Tyr, Pro, and Asn, respectively. The time course using N-acetylglucosamine pentamer as a substrate showed a reduction of the rate constant of glycosidic cleavage and transglycosylation and increase of binding free energy for subsite E, which proved the contribution of amino acids mentioned above for substrate binding at subsites E and F.     

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).     

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.     

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 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.     

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

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

Effects of essential carbohydrate/aromatic stacking interaction with Tyr100 and Phe259 on substrate binding of cyclodextrin glycosyltransferase from alkalophilic Bacillus sp. 1011

The stacking interaction between a tyrosine residue and the sugar ring at the catalytic subsite -1 is strictly conserved in the glycoside hydrolase family 13 enzymes. Replacing Tyr100 with leucine in cyclodextrin glycosyltransferase (CGTase) from Bacillus sp. 1011 to prevent stacking significantly decreased all CGTase activities. The adjacent stacking interaction with both Phe183 and Phe259 onto the sugar ring at subsite +2 is essentially conserved among CGTases. F183L/F259L mutant CGTase affects donor substrate binding and/or acceptor binding during transglycosylation [Nakamura et al. (1994) Biochemistry 33, 9929-9936]. To elucidate the precise role of carbohydrate/aromatic stacking interaction at subsites -1 and +2 on the substrate binding of CGTases, we analyzed the X-ray structures of wild-type (2.0 A resolution), and Y100L (2.2 A resolution) and F183L/F259L mutant (1.9 A resolution) CGTases complexed with the inhibitor, acarbose. The refined structures revealed that acarbose molecules bound to the Y100L mutant moved from the active center toward the side chain of Tyr195, and the hydrogen bonding and hydrophobic interaction between acarbose and subsites significantly diminished. The position of pseudo-tetrasaccharide binding in the F183L/F259L mutant was closer to the non-reducing end, and the torsion angles of glycosidic linkages at subsites -1 to +1 on molecule 1 and subsites -2 to -1 on molecule 2 significantly changed compared with that of each molecule of wild-type-acarbose complex to adopt the structural change of subsite +2. These structural and biochemical data suggest that substrate binding in the active site of CGTase is critically affected by the carbohydrate/aromatic stacking interaction with Tyr100 at the catalytic subsite -1 and that this effect is likely a result of cooperation between Tyr100 and Phe259 through stacking interaction with substrate at subsite +2.     

Bare-faced curassow lysozyme carrying amino acid substitutions at subsites E and F shows a change in activity against chitooligosaccharide caused by a local conformational change

A new form of avian lysozyme, bare-faced curassow lysozyme (BCL), was purified and chemically sequenced. Of the 26 substitutions relative to chicken lysozyme, three, F34Y, T47S, and R114H, are of substrate-interacting residues in the E and F subsites, which would contribute to the acceptor binding for transglycosylation. T47S is a novel substitution in this lysozyme class. While other lysozymes also have substitutions at positions 114 and 34, they also contain numerous others, including ones in the other substrate binding sites, A-D. Furthermore, T47S lies on the left side, while F34Y and R114H are located on the right side of the E-F subsites. BCL therefore should allow comparison of the independent contributions of these sites to substrate binding and transglycosylation. The activity toward the N-acetylglucosamine pentamer revealed that the substitutions at the E-F sites reduced the binding free energies at the E-F sites and the rate constant for transglycosylation without the conformation change of other substrate binding sites on the protein. MD simulation analysis of BCL suggested that the substituted amino acids changed the local conformation of this lysozyme at the E-F sites.     

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.     

Subsite mapping of Aspergillus niger endopolygalacturonase II by site-directed mutagenesis

To assess the subsites involved in substrate binding in Aspergillus niger endopolygalacturonase II, residues located in the potential substrate binding cleft stretching along the enzyme from the N to the C terminus were subjected to site-directed mutagenesis. Mutant enzymes were characterized with respect to their kinetic parameters using polygalacturonate as a substrate and with respect to their mode of action using oligogalacturonates of defined length (n = 3-6). In addition, the effect of the mutations on the hydrolysis of pectins with various degrees of esterification was studied. Based on the results obtained with enzymes N186E and D282K it was established that the substrate binds with the nonreducing end toward the N terminus of the enzyme. Asn(186) is located at subsite -4, and Asp(282) is located at subsite +2. The mutations D183N and M150Q, both located at subsite -2, affected catalysis, probably mediated via the sugar residue bound at subsite -1. Tyr(291), located at subsite +1 and strictly conserved among endopolygalacturonases appeared indispensable for effective catalysis. The mutations E252A and Q288E, both located at subsite +2, showed only slight effects on catalysis and mode of action. Tyr(326) is probably located at the imaginary subsite +3. The mutation Y326L affected the stability of the enzyme. For mutant E252A, an increased affinity for partially methylesterified substrates was recorded. Enzyme N186E displayed the opposite behavior; the specificity for completely demethylesterified regions of substrate, already high for the native enzyme, was increased. The origin of the effects of the mutations is discussed.     

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.     

Transglycosylation reaction catalyzed by a class V chitinase from cycad, Cycas revoluta: A study involving site-directed mutagenesis,HPLC, and real-time ESI-MS

Class V chitinase from cycad, Cycas revoluta, (CrChi-A) is the first plant chitinase that has been found to possess transglycosylation activity. To identify the structural determinants that bring about transglycosylation activity, we mutated two aromatic residues, Phe166 and Trp197, which are likely located in the acceptor binding site, and the mutated enzymes (F166A, W197A) were characterized. When the time-courses of the enzymatic reaction toward chitin oligosaccharides were monitored by HPLC, the specific activity was decreased to about 5–10% of that of the wild type and the amounts of transglycosylation products were significantly reduced by the individual mutations. From comparison between the reaction time-courses obtained by HPLC and real-time ESI-MS, we found that the transglycosylation reaction takes place under the conditions used for HPLC but not under the ESI-MS conditions. The higher substrate concentration (5 mM) used for the HPLC determination is likely to bring about chitinase-catalyzed transglycosylation. Kinetic analysis of the time-courses obtained by HPLC indicated that the sugar residue affinity of + 1 subsite was strongly reduced in both mutated enzymes, as compared with that of the wild type. The IC₅₀ value for the inhibitor allosamidin determined by real-time ESI-MS was not significantly affected by the individual mutations, indicating that the state of the allosamidin binding site (from − 3 to − 1 subsites) was not changed in the mutated enzymes. We concluded that the aromatic side chains of Phe166 and Trp197 in CrChi-A participate in the transglycosylation acceptor binding, thus controlling the transglycosylation activity of the enzyme.     

Additional binding sites in lysozyme. X-ray analysis of lysozyme complexes with bromophenol red and bromophenol blue.

The binding sites in hen egg-white lysozyme for neutral bromophenol red (BPR) and ionized bromophenol blue (BPB) have been characterized at 2 A resolution. In either case, the dye-bound enzyme is active against the polysaccharide, but not against the cell wall. Both binding sites are outside, but close to, the hexasaccharide binding cleft in the enzyme. The binding site of BPR made up of Arg5, Lys33, Phe34, Asn37, Phe38, Ala122, Trp123 and possibly Arg125, is close to subsite F while that of BPB made up of Tyr20, Arg21, Asn93, Lys96, Lys97 and Ser100, is close to subsites A and B. The binding sites of the neutral dye and the ionized dye are thus spatially far apart. The peptide component of the bacterial cell wall probably interacts with these cells during enzyme action. Such interactions are perhaps necessary for appropriately positioning the enzyme molecule on the bacterial cell wall.     

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.     

Complete amino acid sequence of three reptile lysozymes

To study the structure and function of reptile lysozymes, we have reported their purification, and in this study we have established the amino acid sequence of three egg white lysozymes in soft-shelled turtle eggs (SSTL A and SSTL B from Trionyx sinensis, ASTL from Amyda cartilaginea) by using the rapid peptide mapping method. The established amino acid sequence of SSTL A, SSTL B, and ASTL showed substitutions of 43, 42, and 44 residues respectively when compared with the HEWL (hen egg white lysozyme) sequence. In these reptile lysozymes, SSTL A had one substitution compared with SSTL B (Gly126Asp) and had an N-terminal extra Gly and 11 substitutions compared with ASTL. SSTL B had an N-terminal extra Gly and 10 residues different from ASTL. The sequence of SSTL B was identical to soft-shelled turtle lysozyme from STL (Trionyx sinensis japonicus). The Ile residue at position 93 of ASTL is the first report in all C-type lysozymes. Furthermore, amino acid substitutions (Phe34His, Arg45Tyr, Thr47Arg, and Arg114Tyr) were also found at subsites E and F when compared with HEWL. The time course using N-acetylglucosamine pentamer as a substrate exhibited a reduction of the rate constant of glycosidic cleavage and increase of binding free energy for subsites E and F, which proved the contribution for amino acids mentioned above for substrate binding at subsites E and F. Interestingly, the variable binding free energy values occurred on ASTL, may be contributed from substitutions at outside of subsites E and F.     

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.     

Identification of acceptor substrate binding subsites +2 and +3 in the amylomaltase from Thermus thermophilus HB8

Glycoside hydrolase family 77 (GH77) belongs to the alpha-amylase superfamily (Clan H) together with GH13 and GH70. GH77 enzymes are amylomaltases or 4-alpha-glucanotransferases, involved in maltose metabolism in microorganisms and in starch biosynthesis in plants. Here we characterized the amylomaltase from the hyperthermophilic bacterium Thermus thermophilus HB8 (Tt AMase). Site-directed mutagenesis of the active site residues (Asp293, nucleophile; Glu340, general acid/base catalyst; Asp395, transition state stabilizer) shows that GH77 Tt AMase and GH13 enzymes share the same catalytic machinery. Quantification of the enzyme's transglycosylation and hydrolytic activities revealed that Tt AMase is among the most efficient 4-alpha-glucanotransferases in the alpha-amylase superfamily. The active site contains at least seven substrate binding sites, subsites -2 and +3 favoring substrate binding and subsites -3 and +2 not, in contrast to several GH13 enzymes in which subsite +2 contributes to oligosaccharide binding. A model of a maltoheptaose (G7) substrate bound to the enzyme was used to probe the details of the interactions of the substrate with the protein at acceptor subsites +2 and +3 by site-directed mutagenesis. Substitution of the fully conserved Asp249 with a Ser in subsite +2 reduced the activity 23-fold (for G7 as a substrate) to 385-fold (for maltotriose). Similar mutations reduced the activity of alpha-amylases only up to 10-fold. Thus, the characteristics of acceptor subsite +2 represent a main difference between GH13 amylases and GH77 amylomaltases.     

Roles of the N-terminal domain and remote substrate binding subsites in activity of the debranching barley limit dextrinase

Barley limit dextrinase (HvLD) of glycoside hydrolase family 13 is the sole enzyme hydrolysing α-1,6-glucosidic linkages from starch in the germinating seed. Surprisingly, HvLD shows 150- and 7-fold higher activity towards pullulan and β-limit dextrin, respectively, than amylopectin. This is investigated by mutational analysis of residues in the N-terminal CBM-21-like domain (Ser14Arg, His108Arg, Ser14Arg/His108Arg) and at the outer subsites +2 (Phe553Gly) and +3 (Phe620Ala, Asp621Ala, Phe620Ala/Asp621Ala) of the active site. The Ser14 and His108 mutants mimic natural LD variants from sorghum and rice with elevated enzymatic activity. Although situated about 40 Å from the active site, the single mutants had 15–40% catalytic efficiency compared to wild type for the three polysaccharides and the double mutant retained 27% activity for β-limit dextrin and 64% for pullulan and amylopectin. These three mutants hydrolysed 4,6-O-benzylidene-4-nitrophenyl-6³-α-d-maltotriosyl-maltotriose (BPNPG3G3) with 51–109% of wild-type activity. The results highlight that the N-terminal CBM21-like domain plays a role in activity. Phe553 and the highly conserved Trp512 sandwich a substrate main chain glucosyl residue at subsite +2 of the active site, while substrate contacts of Phe620 and Asp621 at subsite +3 are less prominent. Phe553Gly showed 47% and 25% activity on pullulan and BPNPG3G3, respectively having a main role at subsite +2. By contrast at subsite +3, Asp621Ala increased activity on pullulan by 2.4-fold, while Phe620Ala/Asp621Ala retained only 7% activity on pullulan albeit showed 25% activity towards BPNPG3G3. This outcome supports that the outer substrate binding area harbours preference determinants for the branched substrates amylopectin and β-limit dextrin.     

Dissection of the functional role of structural elements of tyrosine-63 in the catalytic action of human lysozyme.

The functional role of tyrosine-63 in the catalytic action of human lysozyme (EC 3.2.1.17) has been probed by site-directed mutagenesis. In order to identify the role of Tyr63 in the interaction with substrate, both the three-dimensional structures and the enzymatic functions of the mutants, in which Tyr63 was converted to phenylalanine, tryptophan, leucine, or alanine, have been characterized in comparison with those of the wild-type enzyme. X-ray crystallographical analysis of the mutant enzyme at not less than 1.77-A resolution indicated no remarkable change in tertiary structure except the side chain of 63rd residue. The conversion of Tyr63 to Phe or Trp did not change the enzymatic properties against the noncharged substrate (or substrate analogs) largely, while the conversion to Leu or Ala markedly reduced the catalytic activity to a few percent of wild-type enzyme. Kinetic analysis using p-nitrophenyl penta-N-acetyl-beta-(1----4)-chitopentaoside (PNP-(GlcNAc)5) as a substrate revealed that the reduction of activity should mainly be attributed to the reduction of affinity between enzyme and substrate. The apparent contribution of the phenolic hydroxyl group and the phenol group in the side chain of Tyr63 was estimated to 0.4 +/- 0.4 and 2.5 +/- 0.8 kcal mol-1, respectively. The result suggested that the direct contact between the planar side-chain group of Tyr63 and the sugar residue at subsite B is a major determinant of binding specificity toward a electrostatically neutral substrate in the catalytic action of human lysozyme.