1 Graduate School of Pharmaceutical Sciences, Kyushu University, Fukuoka 8128582, 2 Department of Bioengineering, Faculty of Engineering, Kagoshima University, Korimoto 8900065 and 3 Central Laboratories for Key Technology, Kirin Brewery Co. Ltd, 1135, Fukuura, Kanazawa-ku, Yokohama 236, Japan
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Keywords: catalyst redesign/glycosidase/glycosyl adduct/intermediate
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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., 1992a). 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., 1991
). We previously reported (Kuroki et al., 1997
) 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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The mutant lysozyme cDNAs used here were prepared with site-directed mutagenesis as reported previously (Inoue et al., 1992a; Hashimoto et al., 1996
). 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., 1992a
).
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., 1992). The emission of lysozyme solution (~20 µM) excited by radiation of 290 nm was scanned from 290 to 400 nm with different concentrations (20500 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., 1985; Ito et al., 1994
). 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 29) at 40°C. The buffers used were 0.02 M acetateHCl (pH 25.5) and 0.02 M TrisHCl (pH 69), 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 2060% 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 acetateHCl (pH 4.0) and eluted with a 01 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., 1992b). The change in the fluorescence intensity of 2 µM lysozyme solution was traced in the pH range 28. 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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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., 1997). 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., 1997
). 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 1.0x103 min1.
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 104 min1 at 50°C and 3.3x105 min1 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 I.
|
The substrate-binding ability of the mutant lysozyme and its covalent adduct prepared here was examined (Table II). 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 II
. 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 II
). This indicates that the covalently attached substrate tightly occupies the binding pocket of the enzyme.
|
The suggested catalytic mechanism for D52E lysozyme is shown in Figure 1B. 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.
|
The formation reaction of the covalent adduct was examined at pH 28. The change in the rate constant with pH is shown in Figure 2A. 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 III
) under the assumed mechanism (Figure 1B
) was in good agreement with the experimental data, supporting the proposed mechanism.
|
|
Design of a secondary mutation for enzymatic improvement
The hydrolysis of the covalent adduct was slower than the formation reaction (Table I). To enhance the turnover rate of D52E lysozyme by accelerating the hydrolysis rate, a mutation of N46D was designed (Figure 1C
). Asn46 is a key residue for both the substrate binding and the catalytic function, although it is not a catalytic residue (Inoue et al., 1992c
; Matsumura and Kirsch, 1996b
). 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 (1.4x103 min1) than that of D52E lysozyme (1.9x103 min1) (Table I). 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 I
). 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 II
).
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 3A. 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 III
), 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 3A
). 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).
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 3A). 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 III
), 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 1C). 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 3B
). 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 acidbase catalyst Glu127 and the nucleophile Glu233. The X-ray crystallographic structure of the covalent adduct with a chemically modified substrate analogue (White et al., 1996
) 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., 1998
). Tyr298 in ß-glycosidase Abg from A.faecalis hydrogen bonds to the catalytic nucleophile Glu358 and also has an equivalent role (Gebler et al., 1995
).
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 (610) of natural glycosidases are suitable for this purpose, because their catalytically active species, protonated forms, predominate below pH 55.5, 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 (3.2x105 min1) through the covalent adduct (Table I) was far less (<1%) than the apparent hydrolysis rate (8x103 min1) 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., 1996
) 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 enzymesubstrate adduct was detected in the reaction of D52S lysozyme and (GlcNAc)6 by electrospray mass spectrometry (Lumb et al., 1992). 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., 1994
). The structure of D52S lysozyme complexed with (GlcNAc)4 also suggested that the distance between the
-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 enzymesubstrate 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 |
---|
![]() |
Notes |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Gebler, J.C., Trimbur, D.E., Warren,A.J., Aebersold, R., Namchuk, M. and Withers, S.G. (1995) Biochemistry, 34, 1454714553.[ISI][Medline]
Hadfield,A.T., Harvey,D.J., Archer,D.B., MacKenzie,D.A., Jeenes,D.J., Radford,S.E., Lowe,G., Dobson,C.M. and Johnson,L.N. (1994 ) J. Mol. Biol., 243, 856872.[ISI][Medline]
Hashimoto,Y., Yamada,K., Motoshima,H., Omura,T., Yamada,H., Yasukochi,T., Miki,T., Ueda,T. and Imoto,T. (1996) J. Biochem., 119, 145150.[Abstract]
Imoto,T., Johnson,L.N., North,A.C.T., Phillips,D.C. and Rupley,J.A. (1972) In Boyer,P.D. (ed.), The Enzyme: Vertebrate Lysozyme. Academic Press, New York, pp. 665836.
Inoue,M., Yamada,H., Yasukochi,T, Kuroki,R., Miki,T., Horiuchi,T. and Imoto,T. (1992a) Biochemistry, 31, 55455553.[ISI][Medline]
Inoue,M., Yamada,H., Yasukochi,T., Miki,T., Horiuchi,T. and Imoto,T. (1992b) Biochemistry, 31, 1032210330.[ISI][Medline]
Inoue,M., Yamada,H., Hashimoto,Y., Yasukochi,T., Hamaguchi,K., Miki,T., Horiuchi,T. and Imoto,T. (1992c) Biochemistry, 31, 88168821.[ISI][Medline]
Ito,Y., Hirashima,M., Yamada,H. and Imoto,T. (1994) J. Biochem., 116, 13461353.[Abstract]
Jolles, P. and Jolles, J. (1984) Mol. Cell. Biochem., 63, 165189.[ISI][Medline]
Karplus,M. and Post,C.B. (1996) EXS, 75, 111141.[Medline]
Kumagai,I., Sunada,F., Takeda,S. and Miura,K. (1992) J. Biol. Chem., 267, 46084612.
Kuroki,R., Ito,Y., Kato,Y., Imoto,T. (1997) J. Biol. Chem., 272, 1997619981.
Lumb,K.J., Aplin,R.T., Radford,S.E., Archer,D.B., Jeenes,D.J., Lambert,N., MacKenzie,D.A., Dobson,C.M. and Lowe,G. (1992) FEBS Lett., 296, 153157.[ISI][Medline]
Malcolm,B.A., Rosenberg,S., Corey,M.J., Allen,J.S., Baetselier,A.D. and Kirsch,J.F. (1989) Proc. Natl Acad. Sci. USA, 86, 133137.[Abstract]
Matsumura,I. and Kirsch,J.F. (1996a) Biochemistry, 35, 18811889.[ISI][Medline]
Matsumura,I. and Kirsch,J.F. (1996b) Biochemistry, 35, 18901896.[ISI][Medline]
Muraki,M., Harata,K., Hayashi,Y., Machida,M. and Jigami,Y. (1991) Biochim. Biophys. Acta, 1079, 229237.[ISI][Medline]
Notenboom, V., Birsan, C., Nitz, M., Rose, D.R., Warren, R.A.J. and Withers, S.G. (1998) Nature Struct. Biol., 5, 812818.[ISI][Medline]
Strynadka,N.C. and James,M.N. (1991) J. Mol. Biol., 220, 401424.[ISI][Medline]
Wang,Q., Trimbur,D., Graham,R., Warren,R.A. and Withers,S.G. (1995) Biochemistry, 34, 1455414562.[ISI][Medline]
White,A., Tull,D., Johns,K., Withers,S.G. and Rose,D.R. (1996) Nature Struct. Biol., 3, 149154.[ISI][Medline]
Yamada, H., Fukumura, T., Ito, Y. and Imoto, T. (1985) Anal. Biochem., 146, 7174.[ISI][Medline]
Received September 4, 1998; revised January 7, 1999; accepted January 10, 1999.