LETTER pubs.acs.org/Langmuir
Kinetic Studies of Dextransucrase Enzyme Reactions on a Substrateor Enzyme-Immobilized 27 MHz Quartz Crystal Microbalance Takanori Nihira,† Toshiaki Mori,*,†,‡,§ Megumi Asakura,† and Yoshio Okahata*,†,§ † ‡
Department of Biomolecular Engineering, Tokyo Institute of Technology, 4259 Nagatsuta, Midori-ku, Yokohama, 226-8501 Japan Japan Science and Technology Agency-Precursory Research for Embryonic Science and Technology (PRESTO)
bS Supporting Information ABSTRACT: Catalytic elongation by dextransucrase (DSase) was monitored directly on a dextran-acceptor- or DSaseimmobilized 27 MHz quartz crystal microbalance (QCM). Kinetic parameters for the binding of the enzyme to the dextran acceptor (kon, koff, and Kd) and enzymatic elongation in the presence of a sucrose monomer (Km for sucrose and kcat) were determined. The kinetic parameters obtained by both methods were consistent.
D
extransucrase (DSase; sucrose:1,6-R-D-glucan-6-R-D-glucosyltransferase [EC 2.4.1.5]) is a glucosyltransferase that catalyzes the transfer of a D-glucose unit from sucrose to a dextran receptor (Scheme 1).1 Notably, this enzyme does not require activated substrates (nucleotide sugars or sugar-1-phosphates) as donors. The characterization and kinetic parameters of DSase have been previously reported.2-4 In addition, a study on applied research has reported the development of immobilized DSase on epoxy-activated acrylic polymers for production of oligosaccharides.5 DSase activity is usually measured by monitoring sucrose hydrolysis to fructose6 or elongation of dextran from radiolabeled sucrose in buffer solution by gel shift assay.4 However, these conventional analyses do not allow the determination of the process kinetics (the binding of the enzyme to the acceptor dextran substrate and to the donor sucrose substrate, as well as the enzymatic elongation rate) in the same device but rather follow exclusively the formation of catalytic products. We have previously reported 27 MHz quartz crystal microbalance (QCM) as a useful tool to directly and quantitatively detect sugar-protein interactions7 and enzyme reactions on polysaccharides,8 such as glucoamylase9 and phosphorylase10 on amylopectin and isomalto-dextranase on dextran.11 The 27 MHz QCM is a very sensitive mass-measuring device that operates in various media such as in vacuum, in the air, and in aqueous solution. Both substrate-enzyme binding and release rate constants (kon and koff, respectively) as well as the hydrolysis rate constant (kcat) can be obtained from time-dependent frequency changes of polysaccharide-immobilized QCM plate.8-11 In these experiments, the polysaccharide substrates were usually r 2011 American Chemical Society
Scheme 1. Illustration of the Reaction Mechanism for Dextran Elongation Catalyzed by DSase
immobilized on the QCM plate in order to avoid denaturation of the enzymes upon immobilization. In this Letter, we report for the first time enzyme reactions of polysaccharides, specifically DSase-catalyzed dextran elongation, on a substrate- or enzyme-immobilized QCM (Scheme 1 and Figure 1). The effect of enzyme immobilization on kinetic Received: November 16, 2010 Revised: January 4, 2011 Published: January 31, 2011 2107
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Langmuir parameters (kon, koff, kcat, and Km) was studied by comparison with the substrate-immobilized QCM method. The dextran-free DSase from a constitutive mutant of Leuconostoc mesenteroides B-512F (strain 16)12 was purchased from
Figure 1. Chemical structures of dextran (200-mer)- and DSaseimmobilized 27-MHz QCM.
LETTER
Wako Pure Chemicals. The dextran-immobilized QCM was prepared as schematized in Figure 1.11 Briefly, 3,30 -dithiodipropionic acid was immobilized on a clean bare Au electrode. Then the carboxylic acid was activated as an N-hydroxysuccinimidyl ester on the surface. Afterward, O-[2-(2-aminooxyethoxy)ethyl]hydroxyamine was reacted with the activated ester, and dextran was anchored through Schiff’s base formation between the reducing end of dextran and the hydroxyamino group by immersing the QCM plate in the aqueous solution. And then the unreacted activated esters were treated with ethanolamine due to the deactivation of the surface. On the other hand, DSase was covalently immobilized onto the QCM plate by an aminecoupling method that uses R-amino-ω-carboxyl poly(ethylene glycol)44 (NH2-PEG44-COOH; MW [PEG44] = 2000 Da) as a spacer to avoid the denaturation of enzymes. An AFFINIX Q4 (Initium Co. Ltd., Tokyo, Japan; http:// www.initium2000.com/) instrument was used as the QCM apparatus (see Supporting Information Figure S1). The instrument contained four 500 μL cells equipped with a 27 MHz QCM plate (8.7 mm diameter quartz plate and 5.7 mm2 area Au electrode) at the bottom of the cell and was coupled to a temperature control system.8-11 The Sauerbrey equation
Figure 2. (A) Reaction scheme and kinetic parameters obtained on the dextran-immobilized QCM. (B) Typical time course of the decrease in frequency (and increase in mass) of the dextran-immobilized QCM in response to addition of DSase, followed by sucrose. The amount of immobilized dextran on a QCM plate was 2.5 pmol cm-2. (C) The effect of different DSase concentrations on enzyme binding. (D) The effect of different sucrose concentrations on the elongation process. The buffer used was 50 mM acetate (pH 5.2) containing 150 mM NaCl and 1 mM CaCl2 (25 °C). 2108
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Table 1. Kinetic Parameters Obtained from Dextran- and DSase-Immobilized QCMa kon (103 M-1 koff (10-3
dextran-immobilized QCM
s-1)
s-1)
89
1.6
Kd
Km
kcat
(nM) (mM) (s-1) 18
dsase-immobilized
3.4
3.5
7.2
3.1
QCM
Obtained kinetic parameters contain (10% experimental errors obtained from at least five times each run. Data were obtained in 50 mM acetate buffer (pH 5.2) containing 150 mM NaCl and 1 mM CaCl2 (25 °C). a
(eq 1) was obtained for the AT-cut shear mode QCM in the air phase, 2F0 2 ΔFair ¼ - pffiffiffiffiffiffiffiffiffi Δm A Fq μq
ð1Þ
reached the steady state at 25 °C. The amount of the immobilized DSase (170 kDa) was calculated to be ca. 10% coverage as a monolayer [85 ( 5 ng (0.5 ( 0.1 pmol) cm-2] from the frequency decrease (mass increase) of 510 ( 10 Hz. This coverage was confirmed by atomic force microscopy (AFM) measurements of the DSase-immobilized QCM surface as shown in Figure S2 in the Supporting Information (diameter of DSase: ca. 6 nm). Figure 2B shows typical frequency changes of the dextranimmobilized QCM as a function of time in response to the addition of DSase and sucrose substrate (50 mM acetate buffer [pH 5.2], 150 mM NaCl, and 1 mM CaCl2; 25 °C). The first frequency decrease (mass increase) indicates binding of the enzyme to the dextran acceptor (ESdex complex formation). When the sucrose monomer was injected after the frequency change reached equilibrium, the frequency gradually decreased (while mass increased) due to elongation of dextran according to the reaction mechanism shown in Scheme 1.8-11 kon
EþSdex f s s ESdex r
where ΔFair is the measured frequency change in the air phase [Hz], F0 is the fundamental frequency of the quartz crystal prior to a mass change [27106 Hz], Δm is the mass change [g], A is the electrode area [0.057 cm2], Fq is the density of quartz [2.65 g cm-3], and μq is the shear modulus of quartz [2.951011 dyn cm-2]. In the air phase, a 0.62 ng cm-2 mass increase per 1 Hz of frequency decrease is expected. However, when the QCM is employed in aqueous solutions, one must consider effects of hydration and/or viscoelasticity of biomolecules (eq 2).13 ΔFwater ¼ -
ΔFwater 2F0 pffiffiffiffiffiffiffiffiffi Δm ΔFair A Fq μq 2
½ESdex ½E½Sdex
ð4Þ
Ka Δmmax ½E0 1þKa ½E0
ð5Þ
Ka ¼ Δm ¼
ð2Þ
We therefore directly calibrated the relationship between ΔFwater and ΔFair of DSase or 200-mer dextran bindings onto a QCM plate. Both ΔFwater and ΔFair values were obtained when DSase with the PEG44 spacer or 200-mer were immobilized on the QCM plate at different amounts (as ΔFair = 200-1000 Hz). There were good linear correlations between ΔFwater and ΔFair (Δm) with a slope of 3.6 ( 0.2 for immobilization of Dsase or a slope of 3.6 ( 0.4 for bindings of dextran oligomers. Thus, frequency decreases (ΔFwater) due to the DSase or dextran oligomer bindings were 3.6 times larger than those in the air phase (ΔFair), because hydrating water vibrates with proteins or dextrans. Therefore, the ΔFwater/ΔFair value for DSase and oligo dextrans was determined to be 3.6 ( 0.4 and the factor of Sauerbrey’s equation (eq 2) for DSase and dextrans in aqueous solutions was obtained as 0.62/3.6 = 0.17 ( 0.02 ng cm-2 per -1 Hz. When the longer dextran (more than 300-mer) was immobilized on the QCM surface, however, ΔFwater was increased exponentially (not linearly) with the increase of ΔFair. Therefore, when the elongation of dextran is followed on the QCM, the initial elongation part (less longer than ca. 300-mer) was used as the initial elongation rate (v0). The noise level of the 27 MHz QCM was (1 Hz in buffer solutions at 25 °C, and the stability of the frequency was (10 Hz for 1 h in buffer at 25 °C. A sensitivity of 0.17 ng cm-2 Hz-1 is large enough to detect the binding of DSase or dextran. The details of these calibration experiments were described previously.13 Each dextran- or enzyme-immobilized QCM cell was filled with 400 μL of 20 mM acetate buffer (pH 5.2) containing 150 mM NaCl and 1 mM CaCl2, and placed until the resonance frequency
ð3Þ
koff
Δmt ¼ Δmmax -f1-expð-t=τÞg
ð6Þ
½ESdex t ¼ ½ESdex max -f1-expð-t=τÞg
ð7Þ
τ-1 ¼ kon ½E0 þkoff
ð8Þ
Binding of DSase (E) to dextran as an acceptor substrate (Sdex) is described by eq 3. We followed the formation of the enzymesubstrate complex (ESdex) on the QCM based on mass changes (Δm). At time t, the mass of ESdex is given by eqs 7 and 8. The relaxation time (τ) associated with enzyme binding is calculated from curve fittings of the QCM frequency decrease during the binding process. When binding experiments were carried out at different concentrations of DSase (0.5-26 nM), the binding and dissociation rate constants (kon and koff) of DSase to the dextran acceptor could be obtained from the slope and intercept of eq 8, respectively, under conditions of [E] . [Sdex]. Curve fittings were performed by using the software of IGOR Pro version 6 with regression analysis. The dissociation constant (Kd) was obtained from the ratio of koff to kon. Figure 2C shows typical time courses for the binding of DSase to the dextran acceptor at some concentrations of enzyme (13, 19.5, and 26 nM). The linear reciprocal plot of the relaxation time (τ) obtained from curve fittings of Figure 2C against DSase concentrations (0.5-26 nM) is shown in Supporting Information Figure S3A. From the slope and intercept of Figure S3A, we obtained binding (kon) and dissociation (koff) rate constants of 89 103 M-1 s-1 and 1.6 10-3 s-1, respectively. A very small dissociation constant (Kd = 18 nM) was obtained from koff/kon. This indicated that DSase binds very strongly to the dextran acceptor. The results are summarized in Table 1. Figure 2D shows the effect of different sucrose concentrations on the elongation process of the ESdex complex. When sucrose 2109
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Figure 3. (A) Reaction schemes and kinetic parameters obtained on the DSase-immobilized QCM. (B) Typical time course of the decrease in frequency (and increase in mass) of the DSase-immobilized QCM in response to addition of dextran, followed by sucrose. The amount of immobilized DSase on a QCM plate was 1.2 pmol cm-2. [Dextran] = 26 nM; [sucrose] = 5 mM. The buffer used was 50 mM acetate (pH 5.2) containing 150 mM NaCl and 1 mM CaCl2 (25 °C).
concentrations increased, the initial elongation rate (vo) also increased. The dextran elongation process catalyzed by DSase is described by the Michaelis-Menten equation (eq 9), where ESdexSsuc is the ternary complex of DSase-dextran-sucrose, Pdex is the product dextran, Pfru is the product fructose, Km is the Michaelis constant for sucrose, and kcat is the elongation rate constant. The ESdex complex was estimated to be 2.5 pmol cm-2 from the frequency change after the binding process shown in Figure 2B at [DSase] = 26 nM in the bulk solution. When the initial velocities (vo) were plotted against sucrose concentrations, the saturation behavior was observed as shown in Figure S3B. The Km for sucrose and kcat values obtained from eq 10 were 3.4 mM and 3.5 s-1, respectively. The results are summarized in Table 1. Km
kcat
ESdex þSsuc f s s ESdex Ssuc f s EPdex þPfru r v0 ¼
vmax ½Ssuc 0 Km þ½Ssuc 0
ð9Þ ð10Þ
The enzyme reaction was also followed using the enzymeimmobilized QCM shown in Figure 3A. When DSase rather than
the dextran acceptor was immobilized on the QCM plate, similar frequency changes were observed in response to addition of the dextran acceptor, followed by the sucrose monomer (Figure 3B). Since the decrease in frequency resulting from binding of the dextran acceptor to DSase was very small because of the small molecular weight of dextran as compared to that of DSase, we did not obtain a precise kinetic analysis for the binding process. When DSase was immobilized directly on the QCM plate without using the PEG44 spacer, the elongation process could not be observed maybe due to the denaturation of DSase on the QCM surface. Figure 3C shows the effect of different sucrose concentrations on the elongation process by the immobilized DSase. When sucrose concentrations increased, the initial elongation rate (v0) also increased. We could discuss only the initial elongation stage, because the relationship between the frequency changes and the mass changes cannot be linear in the longer elongation process. The elongation process is also described by the Michaelis-Menten equation (eq 9), as shown in the Supporting Information (Figure S4). The obtained Km for sucrose and kcat values are summarized in Table 1. The elongation rate constant (kcat) and Km values for sucrose determined by both methods were consistent with each other. 2110
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Langmuir Thus, the enzymatic activity of DSase was not affected by immobilization on the QCM plate. This may be due to the use of a long PEG spacer group (see Figure 1). The Km value of DSase for sucrose monomers has been also obtained to be 3 mM in the bulk solution,2 which was well consistent with our Km values obtained on the QCM (Km = 3.4-7.2 mM). DSase was found to have high affinity to the acceptor dextran as the first substrate (Kd = 18 nM) in comparison to the sucrose monomer as the second substrate (Km = 3.4-7.2 mM). DSase has been reported to have high affinity to the dextran oligomer in the bulk solution.14 After binding to the dextran acceptor (and consequent formation of the ESdex complex), DSase can quickly proceed to elongation (kcat = 3.1-3.5 s-1) because the decomposition rate of the ESdex complex is very small (koff = 0.0016 s-1). These features have been observed specifically in several other elongation enzymes.10,11 In conclusion, the catalytic polymerization of dextransucrase from L. mesenteroides was monitored directly on a dextran-acceptoror enzyme-immobilized 27 MHz QCM. This is the first example showing that kinetic parameters can be determined using both the acceptor substrate- and the enzyme-immobilized QCMs in situ in the same device. We believe that the QCM system is a highly sensitive method of detection for in situ enzymatic reactions on unlabeled polysaccharides.
LETTER
(11) Nihira, T.; Mizuno, M.; Tonozuka, T.; Sakano, Y.; Mori, T.; Okahata, Y. Biochemistry 2005, 44, 9456–9461. (12) Mizutani, N.; Yamada, M.; Takayama, K.; Shoda, M. J. Ferment. Bioeng. 1994, 77, 248–251. (13) (a) Ozeki, T.; Morita, M.; Yoshimine, H.; Furusawa, H.; Okahata, Y. Anal. Chem. 2007, 79, 79–88. (b) Furusawa, H.; Ozeki, T.; Morita, M.; Okahata, Y. Anal. Chem. 2009, 81, 2268–2273. (14) Suwannarangsee, S.; Moulis, C.; P.-Veronese, G.; Monsan, P.; R.-Simeon, M.; Chlalaksananukul, W. FEBS Lett. 2007, 581, 4675–4680.
’ ASSOCIATED CONTENT
bS
Supporting Information. Schematic showing the QCM setup; AFM image of DSase-immobilized QCM surface using a PEG44 linker; linear reciprocal plots of relaxation time versus DSase concentration; Michaelis-Menten saturation curves of initial elongation rate versus sucrose concentration. This material is available free of charge via the Internet at http://pubs.acs.org.
’ AUTHOR INFORMATION Corresponding Author
*(Y.O.) E-mail:
[email protected]. Telephone: þ81-45924-5781. (T.M.) E-mail:
[email protected]. Telephone: þ81-45- 924-5782. Author Contributions §
These authors contributed equally to this work.
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