Analysis of a catalytic pathway via a covalent adduct of D52E hen egg white mutant lysozyme by further mutation

Yuji Ito1,2, Ryota Kuroki3, Yoko Ogata1, Yoshio Hashimoto1, Kazuhisa Sugimura2 and Taiji Imoto1,4

1 Graduate School of Pharmaceutical Sciences, Kyushu University, Fukuoka 812–8582, 2 Department of Bioengineering, Faculty of Engineering, Kagoshima University, Korimoto 890–0065 and 3 Central Laboratories for Key Technology, Kirin Brewery Co. Ltd, 1–13–5, Fukuura, Kanazawa-ku, Yokohama 236, Japan


    Abstract
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 Abstract
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 Materials and methods
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We previously demonstrated by X-ray crystallography and electrospray mass spectrometry that D52E mutant hen lysozyme formed a covalent enzyme–substrate adduct on reaction with N-acetylglucosamine oligomer. This observation indicates that D52E lysozyme may acquire a catalytic pathway via a covalent adduct. To explain this pathway, the formation and hydrolysis reactions of the covalent adduct were investigated. Kinetic analysis indicated that the hydrolysis step was the rate-limiting step, 60-fold slower than the formation reaction. In the formation reaction, the pH dependence was bell-shaped, which was plausibly explained by the functions of the two catalytic pKas of Glu35 and Glu52. On the other hand, the pH dependence in the hydrolysis was sigmoidal with a transition at pH 4.5, which was identical with the experimentally determined pKa of Glu35 in the covalent adduct, indicating that Glu35 functions as a general base to hydrolyze the adduct. To improve the turnover rate of D52E lysozyme, the mutation of N46D was designed and introduced to D52E lysozyme. This mutation reduced the activation energy in the hydrolysis reaction of the covalent adduct by 1.8 kcal/mol at pH 5.0 and 40°C but did not affect the formation reaction. Our data may provide a useful approach to understanding the precise mechanism of the function of natural glycosidases, which catalyze via a covalent adduct.

Keywords: catalyst redesign/glycosidase/glycosyl adduct/intermediate


    Introduction
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 Abstract
 Introduction
 Materials and methods
 Results
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Hen egg white lysozyme (HEWL) is an enzyme that degrades the bacterial cell wall by hydrolyzing the ß-1,4-glycosyl bond of N-acetylmuramic acid and N-acetylglucosamine (GlcNAc) copolymer or of GlcNAc oligomer, chitin (Imoto et al., 1972Go; Jolles and Jolles, 1984Go). This enzyme, which has been studied previously as a model protein for elucidating enzymatic function and protein stability, recently became a target protein for protein engineering. Its catalytic mechanism was originally proposed by Phillips and co-workers on the basis of X-ray crystallographic studies of a model complex between enzyme and substrate (Blake et al., 1967Go). Of the six subsites (A–F) for substrate binding, the glycosyl bond between the sugars of D and E was hydrolyzed by the catalytic actions of Glu35 and Asp52 side chains (Imoto et al., 1972Go; Malcolm et al., 1989Go; Strynadka. and James, 1991Go). Glu35 can function as a general acid–base catalyst to donate a proton to the glycosyl oxygen and to remove a proton from a water molecule attacking the oxocarbonium ion in the transient state. On the other hand, it was suggested that Asp52 stabilizes oxocarbonium ion by interacting electrostatically. However, the catalytic mechanism of this electrostatic interaction and the significance of Asp52 as a catalyst have not been confirmed and are still under discussion (Karplus and Post, 1996Go; Matsumura and Kirsch, 1996aGo).

It has been reported that D52E mutant lysozyme largely loses its activity in spite of a slight decrease in substrate binding ability (Inoue et al., 1992aGo). The reduction of catalytic activity in a mutant of D53E human lysozyme (an equivalent mutant of D52E hen lysozyme) has been explained by spatial deviation of the catalytically important negative charge from the correct positioning critical for catalysis (Muraki et al., 1991Go). We previously reported (Kuroki et al., 1997Go) that D52E mutant formed a covalent adduct on reaction with a substrate (GlcNAc)6. In this work, we examined the kinetics of the formation and hydrolysis of the covalent adduct to clarify its significance in the catalytic reaction of D52E lysozyme. The results indicated that D52E lysozyme shows the same catalytic mechanism as the natural glycosidases catalyzing via a covalent intermediate. To promote the turnover rate of D52E lysozyme by a secondary mutation, an N46D mutation was designed and introduced into D52E lysozyme. The catalytic mechanisms of these mutant lysozymes will be discussed as a model for natural glycosidases.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Preparation of mutant lysozymes

The mutant lysozyme cDNAs used here were prepared with site-directed mutagenesis as reported previously (Inoue et al., 1992aGo; Hashimoto et al., 1996Go). Mutations were confirmed by DNA and amino acid sequencing. Both the secretion of lysozyme in a yeast, Saccharomyces cerevisiae AH22, and the subsequent purification of mutant lysozymes were carried out by ion-exchange chromatography according to the previous report (Inoue et al., 1992aGo).

Binding studies with the substrate

Dissociation constants of the complex between lysozyme and (GlcNAc)4 were determined by a slightly modified fluorescence method (Kumagai et al., 1992Go). The emission of lysozyme solution (~20 µM) excited by radiation of 290 nm was scanned from 290 to 400 nm with different concentrations (20–500 µM) of (GlcNAc)4 at 40°C using a Hitachi F-2000 spectrofluorimeter. The dissociation constants (Kd) were determined from a Scatchard plot by plotting the change in the fluorescence intensity at 315 nm.

The substrate binding to GlcNAc polymer, chitin, was examined by an affinity chromatography on a chitin-coated Celite column according to previous reports (Yamada et al., 1985Go; Ito et al., 1994Go). The binding ability of lysozyme was estimated from the retention time and expressed as a relative ratio to the retention time of hen lysozyme.

Analysis of the covalent adducts by reversed-phase HPLC

Lysozyme (20 µM) and (GlcNAc)6 (1 mM) were incubated in separate buffers (pH 2–9) at 40°C. The buffers used were 0.02 M acetate–HCl (pH 2–5.5) and 0.02 M Tris–HCl (pH 6–9), whose ionic strength was adjusted to 0.1 with NaCl. The reaction mixture was injected into a reversed-phase column (250x4.6 mm i.d., ODS-120T; Tosoh) and eluted with a gradient of 20–60% CH3CN containing 0.1% HCl. The protein eluted was detected by measuring the absorbance at 280 nm.

Purification of the covalent adduct

The covalent adduct from the reaction of lysozyme and substrate was purified by ion-exchange HPLC on a Shodex ES502C column. A mixture of 40 µM lysozyme and 1 mM (GlcNAc)6 was incubated at pH 5.0 and 40°C for 24 h to yield 95 and 70% glycosyl form for D52E and N46D/D52E lysozymes, respectively. The reaction mixture was injected into the column equilibrated with 0.1 M sodium acetate–HCl (pH 4.0) and eluted with a 0–1 M NaCl gradient. The covalent adduct, which was eluted earlier than the authentic form, was collected, immediately acidified to pH < 3 with 1 M acetic acid and subsequently subjected to gel permeation chromatography on Sephadex G-25 with 0.1 M acetic acid. The protein fraction was lyophilized and stored at –20°C until used.

pKa titration of catalytic residue

The pKas of the catalytic residues of Glu35 and Asp/Glu 52 were determined at 20°C by the fluorescence method, as described previously (Inoue et al., 1992bGo). The change in the fluorescence intensity of 2 µM lysozyme solution was traced in the pH range 2–8. Scanning was performed in the presence or absence of 1 mM (GlcNAc)4 for free enzyme or the adduct form, respectively. The formation or hydrolysis of the covalent adducts was checked by reversed-phase HPLC after finishing the experiments, but little change was observed for the duration of the experiment (<90 min).


    Results
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 Materials and methods
 Results
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Formation and hydrolysis of the covalent adduct of D52E lysozyme

D52E lysozyme was reacted with 1 mM (GlcNAc)6 at pH 5.0 and 40°C and the reaction mixture was analyzed by reversed-phase HPLC (Kuroki et al., 1997Go). The chromatogram showed a single peak that was identical with authentic D52E lysozyme at zero time. It changed time-dependently with a decrease in the authentic peak and an increase in a new peak that eluted in front of the authentic peak, which had been identified as the adduct form of D52E lysozyme in our previous study (Kuroki et al., 1997Go). The yield of the covalent adduct reached ~95% of total protein after 24 h. The formation reaction proceeded with first-order kinetics and the rate constant (ka) was estimated to be 1.0x10–3 min–1.

To confirm the reversibility of the glycosylation of D52E lysozyme, the hydrolytic reaction of the D52E lysozyme adduct was examined. The purified adduct was dissolved in 0.1 M acetate buffer (pH 5) at 40 or 50°C and analyzed by reversed-phase HPLC. The adduct of D52E lysozyme was slowly hydrolyzed to the free form with a rate constant (kd) of 1.1x 10–4 min–1 at 50°C and 3.3x10–5 min–1 at 40°C. Thus the glycosylation reaction of D52E lysozyme was reversible, but the hydrolysis reaction was 56-fold slower than the formation reaction at 40°C. The kinetic parameters determined here are summarized in Table IGo.


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Table I. Rate constants of formation (ka) and hydrolysis (kd) reactions of the covalent adduct of D52E and N46D/D52E lysozymes at pH 5.0
 
Substrate-binding properties of the covalent adduct

The substrate-binding ability of the mutant lysozyme and its covalent adduct prepared here was examined (Table IIGo). The dissociation constant (Kd) of the complex between D52E lysozyme and (GlcNAc)4 was 3-fold larger than that of wild-type lysozyme. This reduction in substrate binding was confirmed by affinity chromatography, as shown in Table IIGo. The relative retention time in the affinity column of D52E lysozyme was 0.65, which is lower than that of wild-type lysozyme (1.0), indicating a decrease in binding ability of the mutant lysozyme. The covalent adduct of D52E lysozyme was completely excluded from the affinity column, indicating a loss of substrate-binding ability (Table IIGo). This indicates that the covalently attached substrate tightly occupies the binding pocket of the enzyme.


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Table II. Substrate binding properties of the free and adduct forms of D52E and N46D/D52E lysozymes
 
Mechanism of catalytic pathway and pKa of the catalytic residues

The suggested catalytic mechanism for D52E lysozyme is shown in Figure 1BGo. The protonated Glu35 donated a proton to the oxygen of the glycosyl bond and the oxocarbonium ion generated after leaving the non-reducing sugar was attacked by the ionized carboxylic side chain of Glu52 to form a covalent adduct. The subsequent hydrolysis of the covalent adduct was catalyzed by the ionized Glu35 residue acting as a single base. To confirm the mechanism, the pH dependences of the reactions and the pKas of Glu35 and Glu52 were determined by pKa titration.



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Fig. 1. The proposed catalytic mechanisms of (A) wild-type, (B) D52E and (C) N46D/D52E lysozymes.

 
pH dependence of formation and hydrolysis of the covalent adduct

The formation reaction of the covalent adduct was examined at pH 2–8. The change in the rate constant with pH is shown in Figure 2AGo. The pH dependence was bell-shaped with a maximum rate at pH 5.2. The simulation curve drawn by using the experimentally determined pKas of Glu35 and Glu52 (6.1 and 3.9, respectively) (Table IIIGo) under the assumed mechanism (Figure 1BGo) was in good agreement with the experimental data, supporting the proposed mechanism.



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Fig. 2. pH dependences of the (A) formation and (B) hydrolysis reactions of the covalent adduct of D52E lysozyme. The solid lines indicate the simulation curves drawn assuming the catalytic mechanisms depicted in Fig. 1BGo. The pKa values used for calculation are from Table IIIGo.

 

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Table III. pKa of the catalytic residues in D52E and N46D/D52E lysozymes and the adduct forms
 
On the other hand, the pH dependence of the hydrolysis was sigmoidal, showing a transition at pH 4.8 which was almost identical with the pKa of Glu35 (4.7) of the covalent adduct (Table IIIGo). This confirmed that the hydrolysis of the covalent adduct was catalyzed by the ionized Glu35 acting as a general base (Figure 1BGo).

Design of a secondary mutation for enzymatic improvement

The hydrolysis of the covalent adduct was slower than the formation reaction (Table IGo). To enhance the turnover rate of D52E lysozyme by accelerating the hydrolysis rate, a mutation of N46D was designed (Figure 1CGo). Asn46 is a key residue for both the substrate binding and the catalytic function, although it is not a catalytic residue (Inoue et al., 1992cGo; Matsumura and Kirsch, 1996bGo). The model building of the covalent adduct suggested that the carboxamide on the side chain of Asn46 could enclose the carboxylic ester of Glu52 with the substrate within 4.0 Å. The replacement of Asn46 with Asp gave no steric hindrance to the surrounding atoms. Furthermore, the protonated form of Asp46 could function as a general acid to donate a proton to the carboxylic ester of Glu52. We therefore prepared N46D/D52E lysozyme and examined its catalytic properties.

Formation and hydrolysis of the covalent adduct of N46D/D52E lysozyme

The rate constant in the formation of the covalent adduct was slightly lower (1.4x10–3 min–1) than that of D52E lysozyme (1.9x10–3 min–1) (Table IGo). On the other hand, the rate of hydrolysis of the covalent adduct increased 18-fold at 40°C and 11-fold at 50°C compared with D52E lysozyme (Table IGo). The reduction of the activation energy induced by this mutation was estimated to be –1.8 and –1.5 kcal/mol at 40 and 50°C, respectively (Table IIGo).

pH dependence of N46D/D52E lysozyme

The pH dependence of the formation reaction of the covalent adduct in N46D/D52E lysozyme is shown in Figure 3AGo. Although the overall pH-dependent profile was similar to that of D52E lysozyme, the maximum rate was at pH 4.2, which was slightly shifted to acidic pH compared with D52E lysozyme. The pKas of Glu35 and Glu52 were also determined as 5.5 and 3.4, respectively (Table IIIGo), both of which were lower than that of D52E lysozyme, implicating the change of the electrostatic field by the mutation of N46D. The simulation curve was drawn assuming these two pKas (Figure 3AGo). Below pH 5, the data were well fitted with the simulation curve, supporting the participation of Glu35 and Glu52 in the formation reaction. However, a non-negligible deviation was observed above pH 6 (see Discussion).



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Fig. 3. pH dependences in the (A) formation and (B) hydrolysis reactions of the covalent adduct of N46D/D52E lysozyme. The solid line in (A) indicates the simulation curve drawn assuming the catalytic mechanisms (Fig. 1CGo) on the basis of the determined pKas of Glu35 and Glu52 in the free form (Table IIIGo). The circle and plus sign in (B) represent the data at 50 and 40°C, respectively.

 
The pH-dependent profile of the hydrolysis reaction was considerably altered by the mutation of N46D. The pH dependence was not sigmoidal as with D52E lysozyme but bell-shaped, showing its maximum rate at pH 4.2. This result suggested that the introduced Asp46 actually functioned as an acid catalyst together with Glu35, as expected (Figure 1CGo). The pKa of Glu35 in the adduct form was determined as 4.9, which was close to that of D52E lysozyme (Table IIIGo). Although the pKa of Asp46 was not directly determined by pKa titration, it was considered to be a normal value (4–4.5) from the calculation based on the pH dependence depicted in Figure 3BGo.


    Discussion
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 Materials and methods
 Results
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 References
 
In spite of the low catalytic activity, the catalytic mechanism of D52E lysozyme is almost identical with that of the natural glycosidases such as ß-1,4-glycosidase Cex from Cellulomonas fimi (Notenboom et al., 1998Go), Abg from Agrobacterium faecalis (Wang et al., 1995Go) and so on. The hydrolytic reaction was catalyzed by two carboxylic residues, namely one general acid–base catalyst and the other a nucleophile, and proceeded by two steps: the first step is the glycosylation of the enzyme by attack of substrate after the removal of non-reducing sugar and the second step is its deglycosylation. Furthermore, it is also a common feature that the second step is rate determining. Such analogies indicate that D52E lysozyme is a model enzyme for natural glycosidases that catalyze through the enzyme–substrate adduct. Otherwise, D52E lysozyme was, compared with natural glycosidases, very poor in catalytic efficiency and the stability of its covalent intermediate was extremely high, which allowed the analysis of the precise mechanisms in the different steps of the reaction. The first glycosylation step proceeds in a concerted fashion with the acid catalyst Glu35 and the nucleophile Glu52. Glu35 with an abnormally high pKa (6.1), which was mainly caused by the electrostatic repulsion of Glu52, can be a good proton donor to the labile glycosidic oxygen of the substrate. After removal of the non-reducing oxygen, the oxocarbonium ion-like anomeric carbon of the sugar was attacked by the side chain of Glu52 with slightly lower pKa (3.9) to form the covalent adduct. The enzymatically inactive intermediate thus formed was so stable that we could isolate and characterize it. The pKa of Glu35 in the covalent adduct was almost normal (4.7), indicating that the electrostatic repulsion with Glu52 was removed by the glycosylation of the Glu52 side chain. The second deglycosylation step proceeded with only one functional general base of Glu35, resulting in a sigmoidal pH dependence of the reaction. However, in the cases of both natural glycosidases and N46D/D52E lysozyme, residues other than this base catalyst seemed to be involved in the reaction and the reaction profile is usually more complex, as mentioned below.

A significant deviation between the simulation curve and the experimental data was observed above pH 6 in the glycosyl formation reaction of N46D/D52E lysozyme (Figure 3AGo). It is possible that the introduced Asp46 affects the formation reaction at high pH. The pKa of Asp46 was not directly determined here, but was thought to be abnormally high (e.g. >5) on account of the electrostatic repulsion with the closely contacting Glu52. The pKa of Glu52 decreased from 3.9 to 3.4 on N46D mutation (Table IIIGo), which can lead to an increase in the pKa of Asp46. The ionization of Asp46 affects the electrostatic field around the catalytic center and/or alters the direction or conformation of the Glu52 side chain, probably resulting in effective attachment of the Glu52 side chain to the anomeric center of the sugar. However, an X-ray crystallographic study of N46D/D52E lysozyme and the correct pKa evaluations of Asp46 by NMR spectroscopy are needed for a detailed discussion.

We introduced the N46D mutation on the D52E mutant lysozyme in order to accelerate the second step of the reaction, the hydrolysis of the covalent adduct. The protonated form of the carboxylate side chain of Asp46 can donate a proton to the carboxyl oxygen on the glycosyl ester between the Glu52 side chain and the sugar and assist the hydrolysis of the glycosyl ester (Figure 1CGo). The rate of the hydrolysis reaction successfully increased 18-fold and its pH dependence clearly indicated the participation of the introduced Asp46 residue in the hydrolysis reaction (Figure 3BGo). In natural glycosidases, such assisting residues for hydrolysis of the covalent adduct were identified. For example, ß-glycosidase Cex from C.fimi catalyzes the reaction with the general acid–base catalyst Glu127 and the nucleophile Glu233. The X-ray crystallographic structure of the covalent adduct with a chemically modified substrate analogue (White et al., 1996Go) demonstrated that the carboxylic oxygen of Glu233 residue, which covalently formed the glycosyl ester with substrate, hydrogen bonded with His205, which further formed a hydrogen bond with the Asp235 residue. This hydrogen-bond network can contribute to the hydrolysis of the covalent adduct by facilitating the transfer of the proton from His205 to the carboxylic oxygen of Glu233. His205 can promote the departure of the sugar from the Glu233 side chain, allowing the stabilization of the carboxylate form of Glu233. The catalytic assisting role of His205 was confirmed by a mutagenesis study of His205, resulting in the accumulation of the covalent adduct (Notenboom et al., 1998Go). Tyr298 in ß-glycosidase Abg from A.faecalis hydrogen bonds to the catalytic nucleophile Glu358 and also has an equivalent role (Gebler et al., 1995Go).

In both cases of the natural glycosidases, the assisting residues should be protonated forms in order to function as an acid catalyst donating a proton to the glycosyl ester. His205 and Tyr298 with generally high pKa (6–10) of natural glycosidases are suitable for this purpose, because their catalytically active species, protonated forms, predominate below pH 5–5.5, which is the optimum pH for glycosidase. On the other hand, in our case, Asp with a generally low pKa (4) is a poor catalyst because its protonated form is a minor constituent at such a pH. Hence our choice for the mutagenesis is not the best one.

It should be mentioned how the catalytic pathway via the covalent adduct contributes to the whole catalytic activity of D52E lysozyme. The turnover rate (3.2x10–5 min–1) through the covalent adduct (Table IGo) was far less (<1%) than the apparent hydrolysis rate (8x10–3 min–1) of D52E lysozyme against (GlcNAc)6. This means that a major part of the apparent catalytic activity of D52E lysozyme does not depend on the glycosyl pathway but on other pathways. Glu52 can function as an electrostatic stabilizer like Asp52 in the wild-type enzyme. However, D52S lysozyme with no negative charge at the 52 site (Hashimoto et al., 1996Go) retained more detectable activity (as much as 2% of the wild-type enzyme) than D52E lysozyme (0.7%). This implies that electrostatic stabilization at the 52 site is not necessary for such low activity.

It was reported previously that an enzyme–substrate adduct was detected in the reaction of D52S lysozyme and (GlcNAc)6 by electrospray mass spectrometry (Lumb et al., 1992Go). However, such a species was not seen in the X-ray crystallographic structure of the complex between D52S lysozyme and GlcNAc oligomer (Hadfield et al., 1994Go). The structure of D52S lysozyme complexed with (GlcNAc)4 also suggested that the distance between the {gamma}-oxygen of Ser52 and the anomeric C-1 carbon of the D sugar was too large for them to be covalently linked. The superposition of the structures of D52E lysozyme and of the D52S lysozyme–(GlcNAc)4 complex showed that the C-1 carbon of the D sugar in the D52S complex closely contacted the carboxylic oxygen of the Glu52 side chain, within 2.0 Å. This short distance will be favorable for the stability of the generated glycosyl adduct. However, the close contact produces steric hindrance and induces the unfavorable structural changes in the ground state of the enzyme–substrate complex, which may lead to high activation energy of the transition states, especially in the glycosyl reaction.

Our data could provide a detailed mechanism and the functional species of the catalysts involved in hen mutant lysozymes that catalyze via a covalent adduct. We believe that such a mutant system is valuable for understanding the precise mechanism of natural glycosidases.


    Acknowledgments
 
This research was partially supported by the Ministry of Education, Science, Sports and Culture of Japan with a Grant-in-Aid for Encouragement of Young Scientists, 09772026, 1998.


    Notes
 
4 To whom correspondence should be addressed. Email: imoto{at}imml.phar.kyushu-u.ac.jp Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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Received September 4, 1998; revised January 7, 1999; accepted January 10, 1999.





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