Catalysis by a New Sialidase, Deaminoneuraminic Acid Residue-cleaving Enzyme (KDNase Sm), Initially Forms a Less Stable alpha -Anomer of 3-Deoxy-D-glycero-D-galacto-nonulosonic Acid and Is Strongly Inhibited by the Transition State Analogue, 2-Deoxy-2,3-didehydro-D-glycero-D-galacto-2-nonulopyranosonic Acid, but Not by 2-Deoxy-2,3-didehydro-N-acetylneuraminic Acid*

(Received for publication, August 27, 1996, and in revised form, November 3, 1996)

Takaho Terada Dagger , Ken Kitajima Dagger §, Sadako Inoue , Jennifer C. Wilson par , Adele K. Norton par , David C. M. Kong par , Robin J. Thomson par , Mark von Itzstein par and Yasuo Inoue **

From the Dagger  Department of Biophysics and Biochemistry, Graduate School of Science, University of Tokyo, Hongo-7, Tokyo 113, Japan, the  Institute of Biological Chemistry, Academia Sinica, Nankang, Taipei 115, Taiwan, and the par  Department of Medicinal Chemistry, Victorian College of Pharmacy, Monash University, Parkville, 3052 Victoria, Australia

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES


ABSTRACT

Deaminoneuraminic acid residue-cleaving enzyme (KDNase Sm) is a new sialidase that has been induced and purified from Sphingobacterium multivorum. Catalysis by this new sialidase has been studied by enzyme kinetics and 1H NMR spectroscopy. Vmax/Km values determined for synthetic and natural substrates of KDNase Sm reveal that 4-methylumbelliferyl-KDN (KDNalpha 2MeUmb, Vmax/Km = 0.033 min-1) is the best substrate for this sialidase, presumably because of its good leaving group properties. The transition state analogue, 2,3-didehydro-2,3-dideoxy-D-galacto-D-glycero-nonulosonic acid, is a strong competitive inhibitor of KDNase Sm (Ki = 7.7 µM versus Km = 42 µM for KDNalpha 2MeUmb). 2-Deoxy-2,3-didehydro-N-acetylneuraminic acid and 2-deoxy-2,3-didehydro-N-glycolylneuraminic acid are known to be strong competitive inhibitors for bacterial sialidases such as Arthrobacter ureafaciens sialidase; however, KDNase Sm activity is not significantly inhibited by these compounds. This observation suggests that the hydroxyl group at C-5 is important for recognition of the inhibitor by the enzyme.

Reversible addition of water molecule (or hydroxide ion) to the reactive sialosyl cation, presumably formed at the catalytic site of KDNase Sm, eventually gives rise to two different adducts, the alpha - and beta -anomers of free 3-deoxy-D-glycero-D-galacto-nonulosonic acid. 1H NMR spectroscopic studies clearly demonstrate that the thermodynamically less stable alpha -form is preferentially formed as the first product of the cleavage reaction and that isomerization rapidly follows, leading to an equilibrium mixture of the two isomers, the beta -isomer being the major species at equilibrium. Therefore, we propose that KDNase Sm catalysis proceeds via a mechanism common to the known exosialidases, but the recognition of the substituent at C-5 by the enzyme differs.


INTRODUCTION

3-Deoxy-D-galacto-D-glycero-nonulosonic acid (KDN)1 is a relatively new member of the sialic acid family in which the N-acyl group at C-5 position of N-acylneuraminic acid (Neu5Acyl) is replaced by a hydroxyl group. Following the first report of KDN in 1986 (1), its association with a diverse number of living organisms ranging from bacterial capsular polysaccharide (2) to animal glycoproteins and glycolipids (3-11) has been demonstrated. Interestingly, immunochemical techniques suggested the presence of an oligomeric form of alpha 2right-arrow8-linked KDN in mammalian tissues (10, 11), and the occurrence of KDN residues was confirmed by biochemical and chemical methods (12). A unique feature of KDN-containing glycoconjugates is their complete resistance to the action of the known bacterial and viral exosialidases (1, 5, 9, 13).

Recently, we demonstrated the existence of a new sialidase, which specifically cleaves both synthetic and naturally occurring KDN-alpha -ketosides in a Gram-negative soil bacterium, Sphingobacterium multivorum (14). This enzyme, designated KDNase Sm, catalyzes the hydrolysis of a wide variety of different KDN-ketosidic linkages, e.g. KDNalpha 2right-arrow3Gal, KDNalpha 2right-arrow 6GalNAc, and KDNalpha 2right-arrow8KDN, in a diverse range of oligosaccharides, glycoproteins, and glycolipids as well as the synthetic substrate, 4-methylumbelliferyl KDN (KDNalpha 2MeUmb) (14). KDNase Sm is an inducible enzyme in the periplasm of the bacteria (15). The purified enzyme consists of a single polypeptide chain with molecular mass of 47.5 kDa (15). Significantly, KDNase Sm completely lacks any N-acylneuraminidase activity that releases Neu5Ac and Neu5Gc from a variety of Neu5Ac- and Neu5Gc-containing glycoconjugates (14). Studies from our group and from Li et al. (13) describe the occurrence of "KDN sialidases" that cleave both KDN and N-acylneuraminyl ketosides in fish ovary (16) and liver (13).

Recent kinetic and crystallographic studies have provided information about the molecular mechanism of exosialidase catalysis of sialyl linkages (17-22). This paper describes a quantitative investigation of the equilibria and kinetics involved in KDNase Sm-catalyzed hydrolysis by the steady-state enzyme kinetics and 1H NMR spectroscopy. This process is discussed in both kinetic and thermodynamic terms and compared with other known bacterial sialidases.


EXPERIMENTAL PROCEDURES

Chemicals

KDN and Neu5Gc were prepared as described previously (23). 4-Methylumbelliferyl KDN (KDNalpha 2MeUmb) was kindly provided by Dr. T. G. Warner (Genentech, Inc.). Neu5Ac and 4-methylumbelliferyl Neu5Ac (Neu5Acalpha 2MeUmb) were purchased from Nacalai (Japan). KDN dimer, KDNalpha 2right-arrow8KDN (hereafter (KDN)2), was prepared from KDN-rich glycoprotein as described previously (24). KDN-lactose, KDNalpha 2right-arrow3Galbeta 1right-arrow4Glc, was prepared by digestion of (KDN)GM3 (5) with Rhodococcus sp. ceramide glycanase (Seikagaku Kogyo, Japan) according to the method previously described (9). In brief, (KDN)GM3 (240 µg) was dissolved with sonication in 250 µl of 0.4% Triton X-100, 10 mM sodium acetate (pH 5.2), and incubated with ceramide glycanase (5 milliunits) at 37 °C for 18 h. To this was added 1.25 ml of chloroform/methanol (2:1, v/v) with mixing. The aqueous layer was applied to a Sep-Pak C18 Cartridge equilibrated with methanol/water (1:1, v/v) and eluted with 2 ml of water. The eluate, containing KDN-lactose, was evaporated and used for kinetic experiments. The KDN-lactose thus obtained was pure based on TLC analysis (Si-HPF-Silica Gel TLC plate (J. T. Baker Inc.), chloroform/methanol/0.2% CaCl2 (55:45:10, v/v/v)).

2-Deoxy-2,3-didehydro-N-acetylneuraminic acid (Neu5Ac2en) was commercially available from Boehringer Mannheim (Germany). N-Acetylneuraminate pyruvate-lyase was obtained from Wako Chemicals, Osaka. 2,3-Didehydro-2,3-dideoxy-D-glycero-D-galacto-2-nonulopyranosonic acid (KDN2en), 2,3-didehydro-2,3,4-trideoxy-D-glycero-D-galacto-2-nonulopyranosonic acid (4-deoxy-KDN2en), and 2-deoxy-2,3-didehydro-N-glycolylneuraminic acid (Neu5Gc2en) were chemically synthesized according to literature procedures (25, 27).2

Enzymes

KDNase Sm was isolated and purified from S. multivorum as described previously (15). One unit of enzyme activity is defined as the amount of enzyme required to catalyze the hydrolysis of 1 nmol of KDNalpha 2MeUmb per min at 25 °C. Exosialidases from Arthrobacter ureafaciens and Clostridium perfringens were purchased from Nacalai Co. (Japan) and Sigma, respectively. One unit of the exosialidase activity is defined as the amount of enzyme that releases 1 µmol of Neu5Ac from sialyllactose per min at 37 °C (28).

Kinetics

Initial rates were measured for each substrate in the presence and absence of inhibitors as follows.

KDNalpha 2MeUmb and Neu5Acalpha 2MeUmb

Each of KDNalpha 2MeUmb and Neu5Acalpha 2MeUmb at concentrations ranging from 13.9 to 139 µM was incubated with 19 milliunits of KDNase Sm or 0.020 milliunits of A. ureafaciens or C. perfringens exosialidase in 21.6 µl of reaction mixture (containing 0.1 M Tris/acetic acid buffer (pH 6.0), 0.1 M NaCl, and 1 mg/ml BSA) at 25 °C for 30 min. After incubation, 3.0 ml of 85 mM glycine/carbonate buffer (pH 9.3) was added to the reaction mixture. The released 4-methylumbelliferone was fluorometrically determined as described previously (29). Control incubation was carried out in an identical fashion but without enzyme.

(KDN)2 and KDN-lactose

A reaction mixture (10 µl) containing 82.0-410 µM (KDN)2 or 89.6-766 µM KDN-lactose, 0.152 units of KDNase Sm, 100 mM Tris/acetic acid buffer (pH 6.0), 100 mM NaCl, and 1 mg/ml BSA was incubated at 25 °C for 30 min for (KDN)2 or 1 h for KDN-lactose. The reaction was terminated by adding 10 µl of cold ethanol and then dried using Speed Vac SC 110. To this was added 25 µl of 50 mM potassium phosphate buffer (pH 7.2) containing 0.025 units of N-acylneuraminate pyruvate lyase which was then incubated at 37 °C for 1 h to destroy any free KDN remaining. The amount of remaining (KDN)2 and KDN-lactose was determined by the thiobarbituric acid method as described previously (30, 31). Control incubation was carried out without enzyme.

Km and Vmax values were determined from Lineweaver-Burk plots. Ki values were estimated by Dixon plots.

1H NMR Experiments

The hydrolysis of KDNalpha 2MeUmb by KDNase Sm was monitored by 1H NMR spectroscopy as described previously (21). The reaction conditions were described in the legend for Fig. 3. A spectrum of KDNalpha 2MeUmb, without KDNase Sm, was also acquired under identical spectral conditions for the purpose of providing a "zero time" spectrum.


Fig. 3. Progress of the KDNase Sm reaction monitored by 600 MHz 1H NMR spectroscopy. Spectral data were acquired at the times indicated on the spectra. The reaction was performed with 2.25 mM KDNalpha 2MeUmb in the presence of 19 units of KDNase Sm at 310 K in 0.6 ml of 50 mM deuterated sodium acetate buffer (pD 6.0) containing 0.1 M NaCl and 1 mg/ml BSA.
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RESULTS

Substrate Specificity of KDNase Sm

Under identical conditions to those used in the kinetic runs, KDN was released from all substrates, linearly, within 1 h. Michaelis constants were determined for KDNalpha 2MeUmb, (KDN)2, and KDN-lactose from Lineweaver-Burk plots and are summarized in Table I. The Vmax/Km value for KDNalpha 2MeUmb (0.033 min-1) was approximately 1.5 and 3.3 times greater than the values of (KDN)2 (0.023 min-1) and KDN-lactose (0.0099 min-1), respectively, indicating that KDNalpha 2MeUmb is the best substrate for KDNase Sm.

Table I.

Kinetic analysis of the KDNase Sm using KDNalpha 2MeUmb and (KDN)2


Substrate Km Vmax Vmax/Km

µM µM/min min-1
KDNalpha 2MeUmb 42 1.4 0.033 (1.0)a
(KDN)2 480 11 0.023 (0.70)
KDN-lactose 505 5.0 0.0099 (0.30)

a  Values in parentheses are given relative to KDNalpha 2MeUmb set equal to 1.0.

Effects of 2,3-Didehydro-2,3-dideoxy Derivatives of Sialic Acid on KDNase Activity

KDN2en strongly inhibited the KDNase Sm activity toward KDNalpha 2MeUmb; however, no inhibition was observed for either Neu5Ac2en or Neu5Gc2en as shown in Fig. 1A. Dixon plot analysis, Fig. 1C, showed that KDN2en was a competitive inhibitor of KDNase Sm with a Ki value of 7.7 µM. Notably, this value was smaller than the Km value (42 µM) for KDNalpha 2MeUmb. When (KDN)2 is used as a substrate, KDN2en inhibited the KDNase activity (Fig. 1B) competitively with a Ki = 8.1 µM; however, neither Neu5Ac2en nor Neu5Gc2en had inhibitory effects at concentrations up to 100 µM (Fig. 1A). For both KDNalpha 2MeUmb and (KDN)2 substrates, no inhibition was observed with 4-deoxy-KDN2en at concentrations up to 100 µM (Fig. 1, A and B).


Fig. 1. Effects of KDN2en, 4-deoxy-KDN2en, Neu5Ac2en, and Neu5Gc2en on the KDNase activity. The enzyme activity toward KDNalpha 2MeUmb (A) and (KDN)2 (B) was determined in the absence and presence of KDN2en (bullet ), 4-deoxy-KDN2en (×), Neu5Ac2en (open circle ), and Neu5Gc2en (triangle ) at the indicated concentrations. C shows Dioxin plots for inhibition of KDNase-catalyzed hydrolysis of KDNalpha 2MeUmb by KDN2en. Concentrations of KDNalpha 2Me-Umb used are indicated in the figure.
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Effects of Free Sialic Acid on KDNase Sm Activity

Free KDN had an inhibitory effect on KDNase Sm activity toward KDNalpha 2MeUmb, whereas free Neu5Ac and Neu5Gc had no effect (data not shown). The concentration required for 50% inhibition of KDNase Sm activity, using the experimental conditions described, was 7.1 mM for KDN, compared with 17 µM for KDN2en; therefore inhibition by KDN was significantly weaker than KDN2en.

Effects of 2,3-Didehydro-2,3-dideoxy Derivatives of Sialic Acid on Exosialidases

As previously reported, Neu5Ac2en was a strong inhibitor for exosialidases from A. ureafaciens (Fig. 2A) and C. perfringens (Fig. 2B) (32). Neu5Gc2en showed inhibitory effects on the activity of both enzymes but to a lesser extent than Neu5Ac2en. Interestingly, Neu5Gc2en displayed quite different effects on these two exosialidase activities, with C. perfringens exosialidase being less sensitive to Neu5Gc2en (Fig. 2, A and B). No inhibition was observed for these bacterial enzymes with KDN2en (Fig. 2, A and B) and 4-deoxy-KDN2en (data not shown) at concentrations up to 100 µM.


Fig. 2. Effects of KDN2en, Neu5Ac2en, and Neu5Gc2en on A. ureafaciens (A) and C. perfringens (B) exosialidase activities. The enzyme activity against Neu5Acalpha 2MeUmb was determined in the absence and presence of KDN2en (bullet ), Neu5Ac2en (open circle ), and Neu5Gc2en (triangle ) at the indicated concentrations.
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1H NMR Experiments

Fig. 3 shows a time course reaction of the hydrolysis of the synthetic substrate KDNalpha 2MeUmb catalyzed by KDNase Sm. The progress of the reaction is conveniently monitored by 1H NMR chemical shifts of the H-3 methylene protons of KDNalpha 2MeUmb and its hydrolysis products alpha -KDN and beta -KDN that resonate in a spectral region free of other KDN resonances. The anomeric configuration of the hydrolysis products is easily determined by the chemical shift of the H-3ax and H-3eq protons. The first spectrum at t = 0 min shows the spectral region from 1.4 to 3.0 ppm of KDNalpha 2MeUmb at 310 K without addition of KDNase Sm. In this region of the spectrum the H-3eq and H-3ax protons of KDNalpha 2MeUmb are clearly seen at 2.84 and 1.98 ppm, respectively, and the methyl resonance of the 4-methylumbelliferyl aglycon is at 2.49 ppm.

The remaining spectra show the hydrolysis reaction after the addition of the KDNase Sm. At t = 5 min, resonances at 2.69 and 1.60 ppm are seen which are due to the H-3eq and H-3ax protons, respectively, of the alpha -anomer of KDN. These signals increase in intensity as the time course proceeds, and at the same time the intensity of the H-3 proton signals at 2.84 and 1.98 ppm decrease as KDNalpha 2MeUmb is hydrolyzed. Eventually, after about 36 min hydrolysis of KDNalpha 2MeUmb is complete, and the signals at 2.69 and 1.60 ppm completely disappear. The resonances at 2.69 and 1.60 ppm steadily increase in intensity until 36 min and then decrease steadily after this time. This is due to conversion of the initial reaction product alpha -KDN to beta -KDN. H-3eq and H-3ax protons of beta -KDN are visible at 2.18 and 1.81 ppm, respectively. Isomerization of alpha -KDN continues until final equilibrium values for the anomeric mixture of approximately 90% beta - and 10% alpha -KDN (as determined by integration of the H-3 signals) are established.


DISCUSSION

On the basis of the Vmax/Km values (Table I), the synthetic substrate (KDNalpha 2MeUmb) was demonstrated to be a better substrate for KDNase Sm than the natural substrates, KDNalpha 2right-arrow8KDN and KDNalpha 2right-arrow3Galbeta 1right-arrow4Glc. This is presumably because the synthetic 4-methylumbelliferyl aglycon is a better leaving group than the naturally occurring leaving groups.

KDN2en is a potent competitive inhibitor for the KDNase Sm-catalyzed reaction as revealed by Dixon plots (Fig. 1C). We propose that KDN2en is a transition-state analogue of KDNase Sm because it fulfills the following criteria (33, 34). (i) A transition-state inhibitor binds more tightly to the enzyme than the substrate; (ii) the structure of the inhibitor mimics that of the substrate in the putative transition-state during the enzyme-catalyzed reaction. The Ki values for KDN2en are significantly smaller than the Km values for the substrates used, i.e. KDNalpha 2MeUmb (Ki = 7.7 µM versus Km = 42 µM) and (KDN)2 (Ki = 8.1 µM versus Km = 480 µM), indicating that KDNase Sm binds more favorably to KDN2en than these substrates. By analogy with 2-deoxy-2,3-didehydro-Neu5Acyl derivatives, i.e. Neu5Acyl2en derivatives, for N-acylneuraminidases, KDN2en can be referred to as a transition-state analogue for KDNase Sm. Interestingly, KDNase Sm and exosialidases were strongly inhibited only by their cognate transition-state analogues, indicating that the binding site in each enzyme is complementary and specifically accommodates the C-5 substituent of the substrates and inhibitors and is an important determinant for enzyme specificity. Data supporting this view include the following: (a) Neu5Gc2en had no inhibitory effect on KDNase Sm; however, it exhibited a similar effect on other exosialidase as Neu5Ac2en; and (b) hydrolysis of KDNalpha 2MeUmb by KDNase Sm was inhibited in the presence of KDN but not Neu5Ac or Neu5Gc. These results suggest that the active site of KDNase Sm is unable to accommodate an N-acyl group at C-5 of sialic acid residues. Also our studies with the C-4 deoxygenated KDN2en derivative 4-deoxy-KDN2en, which did not inhibit the KDNase Sm activity, provide evidence for the notion that a hydrogen bond donor or acceptor is required at the C-4 position of KDN2en for enzyme recognition.

The stability of KDN and Neu5Acyl may be attributed to both electronic and steric factors that provide a satisfactory interpretation of the kinetic and equilibrium results. It is likely that the initial step in KDNase- or sialidase-catalyzed hydrolysis of alpha -ketosides involves rapid removal of their aglycon moiety at C-2. An attack on the putative sialosyl cation by a nucleophile such as a water molecule is facilitated by the partial positive charge on C-2. It is therefore convenient to discuss reaction in terms of sialosyl cation (K+/enzyme) formation. From analysis of the experimental results depicted in Fig. 3, it may be concluded that hydration of the unstable intermediate (K+/enzyme) takes place initially from below the plane formed by -COO- group/C-6/Oring/C-2/C-3 to give the product alpha -anomer of free KDN. Formation of the alpha -anomer proceeds rapidly (kalpha  > kbeta ) indicating that the free energy of activation is low compared with the corresponding value associated with the beta -anomer. This lower activation energy may simply be due to steric hindrance around the beta -face of the cation in the active site, providing a complete stereofacial selectivity in favor of the alpha -face. Our mechanistic studies with influenza virus sialidase demonstrated that formation of the alpha -Neu5Ac was a consequence of the steric hindrance to the beta -face of the sialosyl cation (19, 20).

Mutarotation of the alpha -anomer then occurs leading to a final equilibrium mixture of two species comprising of the alpha -isomer (~10%) and thermodynamically preferred beta -isomer (~90%) of KDN. It is well known that the most thermodynamically stable isomer is not always the initial product of a chemical reaction and that less stable isomers may be formed first (26), which is the case for the KDNase Sm-catalyzed hydrolysis. KDNase Sm- or sialidase-catalyzed reversible hydrolysis is represented schematically in Fig. 4.


Fig. 4. Dual reactivity of an enzyme-bound sialosyl cation (K+/enzyme) during KDNase Sm-catalyzed hydrolysis of KDNalpha 2MeUmb. Hydration of this unstable intermediate (K+/enzyme) takes place preferentially from below the plane formed by -COO- group/C-6/Oring/C-2/C-3 to form the alpha -anomer (kalpha  > kbeta ). Under the same conditions, mutarotation of the alpha -anomer occurs to give the beta -anomer in the proportions 10:90%, respectively, at equilibrium.
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In this investigation we have highlighted the similarities and differences of a new class of sialidase (KDNase Sm) and other known bacterial exosialidases. To fully understand the catalytic mechanism of action of KDNase Sm, however, a crystal structure investigation of this sialidase is necessary, which is currently being pursued in our laboratories.


FOOTNOTES

*   This research was supported by the Mizutani Foundation for Glycoscience (to Y. I. and K. K.), Grant-in-aid for General Scientific Research Grant 07680658 (to S. I.), Developmental Scientific Research Grant 07558211 (to K. K.), and the Australian Research Council and the Monash Research Fund (to M. v. I.). The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§   Present address: Dept. of Applied Biological Sciences, School of Agricultural Sciences, Nagoya University, Chikusa, Nagoya 464-01, Japan.
**   To whom correspondence should be addressed. Tel.: 886-2-785-5696 (ext. 6020); Fax: 886-2-7889759; E-mail: syinoue{at}gate.sinica.edu.tw.
1    The abbreviations used are: KDN, 3-deoxy-D-glycero-D-galacto-nonulosonic acid; Neu5Ac, N-acetylneuraminic acid; Neu5Gc, N-glycolylneuraminic acid; Neu5Acyl, N-acylneuraminic acid; KDN-lactose, KDNalpha 2right-arrow3Galbeta 1right-arrow4Glc; (KDN)GM3, KDNalpha 2right-arrow3Galbeta 1right-arrow4Glcbeta 1right-arrowCer; KDNase, deaminoneuraminic acid residue-cleaving enzyme; KDN2en, 2-deoxy-2,3-didehydro-D-glycero-D-galacto-2-nonulopyranosonic acid; Neu5Ac2en, 2-deoxy-2,3-didehydro-N-acetylneuraminic acid; Neu5Gc 2en, 2-deoxy-2,3-didehydro-N-glycolyl-neuraminic acid; 4-deoxy-KDN2en, 2,3-didehydro-2,3,4-trideoxy-D-glycero-D-galacto-2-nonulo-pyranosonic acid; KDNalpha 2MeUmb, 4-methylumbelliferyl KDN; Neu5Acalpha 2MeUmb, 4-methylumbelliferyl Neu5Ac; (KDN)2, KDNalpha 2right-arrow 8KDN; BSA, bovine serum albumin.
2    A. K. Norton, G. K. Kok, and M. von Itzstein, manuscript in preparation.

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