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Lipase Catalysis on Monolayers at the Air/Water Interface. 1. Kinetic Rate Constants on Quasi-Two-Dimension Keiji Tanaka, Patricia A. Manning,† and Hyuk Yu* Department of Chemistry, University of WisconsinsMadison, Madison, Wisconsin 53706 Received July 13, 1999. In Final Form: November 5, 1999 Chemical reactions on a cell membrane surface are of pivotal importance for cellular functions such as intracellular signal transduction. Of the variety of membrane proteins, those imbedded in hemi-leaflets of bilayer membranes can be mimicked structurally and dynamically on monomolecular layers of phospholipids at the air/water interface. This is to report the kinetics of lipase (Pseudomonas cepacia) catalyzed hydrolysis of a substrate (umbelliferone stearate) on uniphasic L-R-dilauroylphosphatidylcholine monolayers at the air/water interface. A novel experimental protocol of the interfacial reaction is employed to probe the enzymatic kinetics. The kinetic rate constants on the monolayers are extracted from the substrate and the enzyme concentration dependences of the initial hydrolysis rate. The lipase on monolayers at the air/water interface is highly activated over that in bulk solution, and the turnover number of the lipase catalysis is strongly dependent on the surface pressure of monolayers.
Introduction The fluid mosaic model of the biomembrane1 is undergoing a serious modification in recent years.2,3 The earlier simple picture of dynamic homogeneity on lateral dimension of hemi-leaflets on the exoplasmic and cytoplasmic sides with asymmetric lipid composition of plasma membranes is no longer tenable. For the lateral heterogeneity on the exoplasmic leaflet, we begin to take note that the microdomains, consisting of complexes of sphingolipids and sphingomyelins with cholesterol, function as rafts for the transport of some membrane constituents or as relay stations in intracellular signal transduction.4 Since the chemistry and kinetics on the leaflets are expected to be influenced by these microdomains, it behooves us to understand first as a benchmark the kinetics of enzymatic catalysis on homogeneous leaflets. Lipid monolayers at the air/water interface are often regarded as a well-represented model of homogeneous exoplasmic leaflets. Studies of monolayers have a long history, starting with Benjamin Franklin’s time,5 and the technique became a pivotal tool in his search to deduce liquid structure by Langmuir6 long before the modern scattering and diffraction methods were available. Investigations of chemical reactions on monolayers followed soon after with oxidation of oleic acid on monolayers with permanganate solution as the substrate,7,8 which was found to depend sensitively on surface lateral pressure; at high surface pressure no oxidation took place because molecules stood up to insulate double bonds from the permanganate subphase. Thus, the subject of chemical reactions on monolayers has been around for quite some time. Since 1970 interfacial lipolytic catalysis on mono†
Permanent address: Epoxylite Corp., Irvine, CA 92713.
(1) Singer, S. J.; Nicolson, G. L. Science 1972, 175, 720. (2) Jacobson, K.; Sheets, E. D.; Simson, R. Science 1995, 268, 1441. (3) Varma, R.; Mayor, S. Nature 1998, 394, 798. (4) Simons, K.; Ikonen, E. Nature 1997, 387, 569. (5) Giles, C. H. Chem. Ind. 1969, November 8, 1616. (6) Langmuir, I. J. Am. Chem. Soc. 1917, 39, 1848. (7) Adam, N. K.; Jessop, G. Proc. R. Soc. London 1926, A112, 362. (8) Alexander, A. E.; Rideal, E. K. Proc. R. Soc. London 1937, A163, 70. (9) Hughes, A. Biochem. J. 1935, 29, 437. (10) Lagocki, J. W.; Boyd, N. D.; Law, J. H.; Ke´zdy, F. J. J. Am. Chem. Soc. 1970, 92, 2923.
layers, first published by Hughes,9 has been studied by several groups,10-14 and especially Verger and co-workers have actively exploited it for many kinds of substrate and enzymes using a trough with two or three reservoirs.12,13 There are at least five reasons for using the monolayer technique for kinetic studies of interfacial enzymes, paraphrasing the list offered by Ransac et al.13 (1) It is possible to transfer the monolayers from one subphase to another, and the “interfacial quality” relative to the molecular orientation, conformation, packing density, dipole density, and lateral viscosity can be varied by means of surface lateral pressure and monolayer composition. (2) The enzyme catalysis reactions are easily followed while monitoring the monolayer parameters outlined above. (3) The barostatic control of monolayers allows the lipid packing density to be maintained the same throughout a reaction course, resulting in minimal perturbations inherent in the accumulation of reaction products. (4) Because of sensitivity of the monolayer technique, it requires very small amounts of lipids and enzyme, crucial for the cases of rare or hard to synthesize lipid and enzymes that are from rare sources or difficult to purify. (5) The effects of inhibitors that are insoluble and surface active analogues of substrates can be probed precisely. Notwithstanding these advantages, there is a dearth of reports on quantitative determinations of kinetic rate constants of lipolytic catalysis on the monomolecular film thus far. Berg et al. attributed this to the fact that the rate-limiting step is often shifted from the interfacial catalytic turnover to some other step such as substrate replenishment or the removal of the product from the microenvironment of the bound enzyme.15 Using enzyme imbedded vesicles, they conclude that the catalytic kinetics is reaction-controlled.15,16 (11) Colacicco, G. Nature 1971, 233, 202. (12) Verger, R.; de Haas, G. H. Annu. Rev. Biophys. Bioeng. 1976, 5, 77. (13) Ransac, S.; Ivanova, M.; Verger, R.; Panaiotov, I. Methods Enzymol. 1997, 286, 263. (14) Momsen, W. E.; Brockman, H. L. Methods Enzymol. 1997, 286, 292. (15) Berg, O. G.; Yu, B.-Z.; Rogers, J.; Jain, M. K. Biochemistry 1991, 30, 7283. (16) Berg, O. G.; Cajal, Y.; Butterfoss, G. L.; Grey, R. L.; Alsina, A.; Yu, B.-Z.; Jain, M. K. Biochemistry 1998, 37, 6615.
10.1021/la9909244 CCC: $19.00 © 2000 American Chemical Society Published on Web 01/13/2000
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Scheme 1
The objective of the present study is to extract kinetic rate constants of the lipase hydrolysis reaction of umbelliferone stearate, newly synthesized amphiphilic substrate, on the L-R-dilauroylphosphatidylcholine (DLPC) monolayer in a fluid state at the air/water interface. Lipases belong to a class of lipolytic enzyme that is the most active at the lipid/water interface, has a wide variety of functions in fat metabolism through its esterase activity for hydrolysis of triglycerides, and is used as drugs against digestive disorders and pancreatic diseases.17
Figure 1. Successive steps of the experimental procedure. The amphiphiles with one and two tails represent UMB-C18 and DLPC, respectively, and lipase is depicted by blocks. The shaded squares denote the barrier. See text for each step. Scheme 2
Experimental Section Materials. DLPC was purchased from Avanti Polar Lipids. DLPC was stored at 253 K just prior to use and then used without further purification after thawing at room temperature for more than 1 h. Solutions with concentration of 0.1-0.2 mM were prepared with HPLC grade chloroform (Aldrich). Phosphate buffer solutions at pH of 7.0, which are composed of 9.13 × 10-2 M dibasic anhydrous sodium phosphate, 3.87 × 10-2 M monobasic sodium phosphate, and 0.1 M sodium chloride, were used as the subphase of monolayers. Water used here was the house deionized, further purified by a Milli-Q system with the initial registivity of greater than 17 MΩ. Lipase Purification. Crude lipoprotein lipase LPL 200S, which is mixture of glycine and lipase, from Pseudomonas cepacia was obtained from Amano International Enzyme Co. For purification, 50 mg of this mixture was dissolved in 4 mL of modified McIlvane buffer in a conical flask. Modified McIlvane buffer, adjusted to pH 8.0 with 0.1 M sodium hydroxide aqueous solution, consists of 0.115 M dibasic sodium phosphate and 1.31 × 10-2 M citric acid in Millipore water. The solution was swirled gently for 1-2 min until all the lipase mixture dissolved, then the products were separated by size exclusion chromatography on a Sephadex G-25 fine gel column (Pharmacia Fine Chemicals). Since the molecular weights of lipase and glycine are approximately 33k18 and 75.1, both are well separated by this method. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis of the separated lipase demonstrates that this method yields lipase which is 100% pure.18 The percent yield for this purification process was typically 40-50%. This is in agreement with the fact that the unpurified lipase mixture contains approximately 50% glycine by weight. The lipase solution was stored at 253 K to preserve enzyme activity. The enzyme has been shown to remain stable under these conditions for a minimum of 3 months.19 Synthesis of Umbelliferone Stearate. The surface active substrate, umbelliferone stearate (UMB-C18), was synthesized from umbelliferone (UMB, 1) and stearic anhydride (2) by a straightforward, one-step reaction as shown in Scheme 1. UMB and stearic anhydride were purchased from Molecular Probes and Sigma, respectively. 4-N,N-Dimethylaminopyridine (DMAP) was from Aldrich, and triethylamine (Et3N) and dichloromethane (17) Schmid, R. D.; Verger, R. Angew. Chem., Int. Ed. Engl. 1998, 37, 1608. (18) Rubingh, D. Unpublished results. (19) Roberts, G. A.; Tombs, M. P. Biochim. Biophys. Acta 1987, 902, 327.
were also obtained from Aldrich and dehydrated with calcium hydride. Stearic anhydride (1.0 equiv), UMB (1.1 equiv), and DMAP (0.3 equiv) were placed in a round-bottom flask. Dichloromethane was used as a solvent to give a final concentration of approximately 0.5 M. The reaction was carried out for 1 h with stirring under argon atmosphere. The reaction mixture was washed with 1 M hydrochloric acid solution to remove Et3N and then washed with saturated sodium bicarbonate aqueous solution to make excess UMB water soluble. The desired ester product remains in the organic phase. Next, two washes with the saturated sodium chloride solution were used to predry the organic layer. UMB-C18 (3) was then precipitated from boiling ethanol. Final products were analyzed by thin layer liquid chromatography under an ultraviolet lamp, nuclear magnetic resonance, and mass spectroscopy. Under the conditions, the reaction gave nearly quantitative yield. Although UMB-C18 itself is not fluorescent, hydrolysis of fatty acid-phenolic ester by lipase gives rise to a fluorogenic phenoxide (4), UMB, as shown in Scheme 2. Solutions with concentration of 1.5-2.5 mM for the hydrolysis reaction were prepared with HPLC grade acetone (Aldrich). Monolayer Preparation and Kinetic Protocols. Figure 1 shows our experimental protocol for the enzymatic catalysis on monolayers, consisting of six distinct steps. The Langmuir trough made of Teflon has two compartments20 with an area of 67.4 cm2 and a volume of 350 mL each, which was housed within an acrylate box for humidity control. The relative humidity within the box was kept at 70% or above, and the temperature was controlled at 296.2 ( 0.5 K during all measurements. The surface tensions of the bare and monolayer-covered surfaces were determined by the Wilhelmy technique using a sandblasted platinum plate. The precision of Π determination is better than 0.05 mN‚m-1. The surface tension γ was determined as a function of time by a Cahn electrobalance until the time dependence dγ/ dt reached approximately 10-3 mN‚(m‚s)-1, which was our operational definition of an apparent equilibrium state. First, DLPC monolayers being in an apparent equilibrium state with the surface pressure range of 5.0-20.0 mN‚m-1 were prepared in the right-side trough compartment by the successive addition method (step 1). The initial surface pressure Πini of the DLPC monolayers was adjusted to obtain a different lipase (20) Verger, R.; de Haas, G. H. Chem. Phys. Lipids 1973, 10, 127.
Lipase Catalysis on Monolayers
Figure 2. Block diagram of experimental setup used in analysis of lipase kinetics at the air/water interface. The white and black rectangles on the trough represent the Wilhelmy plate and barriers, respectively. The system is mainly composed of two circuits, the fluorescence detection and the surface pressure control. surface concentration on the monolayer after a lipase solution injection into the subphase. A lipase solution was injected beneath the monolayer to obtain the total lipase concentration of 1.0 × 10-8 M in bulk, and an equilibrium partitioning of the lipase was allowed to reach, taking approximately 1 h (step 2). Since the equilibrium surface pressure Πeq after lipase adsorption to the DLPC monolayer was not sensitive to the Πini and its values were in the range of 25-27 mN‚m-1, it was possible to regulate the lipase surface concentration by changing the Πini of the DLPC monolayer. At this stage, lipase existed not only on the monolayer but also in the subphase and on the Teflon wall. We then transferred the monolayer to the left-side compartment with the aid of a pair of Teflon barriers with speed of 0.13 mm‚s-1 under the condition of the constant surface pressure being at Πeq. The purposes were to isolate the preformed, enzyme-imbedded lipid monolayer from possible effects of lipase in bulk subphase on the interfacial hydrolysis (steps 3 and 4). Once the transfer was completed, the desired surface pressure was set by moving a Teflon barrier. Then, hydrolysis reaction of UMB-C18 was started by injecting its solution into the water subphase, which was being gently stirred by a magnetic stirring bar (step 5). At pH 7.0, UMB-C18 is spontaneously and promptly adsorbed to the monolayer and gets hydrolyzed by the lipase, and the hydrolysis products, stearate and fluorogenic phenoxide formed of umbelliferone, spontaneously desorb into the subphase (step 6). A block diagram of our experimental setup is displayed in Figure 2. Driven by a peristaltic pump, the subphase of the leftside compartment flows through a spectrophotometric cell placed in a fluorometer (MK2, Optical Technology Devices). The excitation and emission wavelengths were fixed at values corresponding to the maximum peak positions, 367 and 454 nm, respectively. Since umbelliferone fluorescence is monitored continuously throughout the reaction period, we claim that a reaction product is monitored in situ.
Results and Discussion Monolayer Transfer. Our purpose is to examine kinetics of enzymatic hydrolysis only on monolayers at the air/water interface. Hence, the lipid monolayer containing lipase was carefully transferred to the second reservoir, as shown in Figure 1, so that any contribution toward the lipase catalysis from those in bulk and/or on the Teflon wall could be minimized. At this point, a specific effort was made to confirm that the desorption of lipase after transfer of monolayer to the second compartment did not take place or was at an undetectable level if it did. The transfer experiment was carried out by using a monolayer with lipase labeled with fluorescein isothiocyanate isomer I (FITC). The fluorescence in the subphase of the left-side compartment was continuously monitored after the monolayer transfer. The monolayer transfer and retention were carried out at Π ) 25 mN‚m-1. A distinct change of the fluorescence intensity could not be observed upon the monolayer transfer and during a period of 1 h after the transfer. The fluorescence intensity change was found to be negligible when the surface pressure was changed within a Π range of 15-25 mN‚m-1. Hence, it is
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altogether probable that lipase desorption does not occur upon the monolayer transfer at the Π range of 15-25 mN‚m-1 for at least 1 h. This result is consistent with the fact of nondesorption of lipid-stabilized lipase in monolayers, established long ago by Verger and co-workers.21 This is central to our claim that only the interfacial hydrolysis reaction is detected in our experiment without any background contribution from the bulk hydrolysis effect. Surface Concentration of UMB-C18. In our experiment, the substrate is injected into the monolayer subphase; therefore the controlling valuable is its bulk concentration. Although knowing the substrate bulk concentration is sufficient to analyze kinetics of the lipase catalysis on monolayers, it is difficult to gain insight into the mechanism of interfacial hydrolysis from bulk concentration alone. Hence, the partitioning of the substrate, UMB-C18, from the bulk subphase to the DLPC monolayer is examined. The widely used experimental technique for determining the surface number density of molecules at the air/water interface is a radioactive tracer method.22 However, we determine the substrate concentration in mixed monolayers based on the surface pressure measurement. Our scheme is not a foolproof way, but perhaps the only way, unless we embark on a study of labeled moiety adsorption and correlating it with the surface pressure, which we did not perform. First, the surface pressure vs area (Π-A) measurement of UMB-C18 itself was carried out by the continuous compression method at the compression speed of 0.13 mm‚s-1 to estimate how much area a UMB-C18 molecule occupies at the air/water interface. The Π-A isotherm of UMB-C18 did not show any signature for phase transition, quite similar to that of stearic acid at room temperature, which has the same alkyl chain length but carboxyl group as the polar head instead of the UMB portion. It exhibits the usual collapse signature at a small area. The limiting area and the collapse pressure obtained from the Π-A isotherm were 0.24 nm2‚molecule-1 and 25.7 mN‚m-1, respectively. The limiting area is determined by the customary extrapolation to Π ) 0 mN‚m-1 of the straight portion of the Π-A curve, and corresponds to the hypothetical cross-sectional area per molecule at Π ) 0 mN‚m-1. Next, the surface pressure change of the monolayer was examined after UMB-C18 solution was injected beneath the DLPC monolayers at Π ) 20 or 25 mN‚m-1. Since it is known how the surface area of DLPC depends on the surface pressure,23 the UMB-C18 surface concentration can be inferred from ∆Π. We here assume that the area per molecule of UMB-C18 at the air/water interface remains at 0.24 mN‚m-1 in DLPC monolayers. Figure 3 shows the relation between substrate bulk concentration, [UMB-C18], and the corresponding one, [UMB-C18]σ, in the DLPC monolayers at Π ) 20 and 25 mN‚m-1. The surface concentration monotonically increases with the amount injected, obeying the Gibbs equation.22 The partitioning of the UMB-C18 from the bulk phase to the monolayer is not large despite its surface activity, presumably by the fact that it competes against DLPC already on the surface. For instance, at Π ) 25 mN‚m-1 and [UMB-C18] ) 100 nM, the surface concentration [UMB-C18]σ is 1.97 × 10-2 molecule‚nm-2. On the other hand, at Π ) 20 mN‚m-1 and the same bulk (21) Rietsch, J.; Pattus, F.; Desnuelle, P.; Verger, R. J. Biol. Chem. 1977, 252, 4313. (22) Adamson, A. W.; Gast, A. P. Physical Chemistry of Surfaces; Wiley-Interscience Publication: New York, 1997; p 80. (23) Tanaka, K.; Manning, P. A.; Lau, V. K.; Yu, H. Langmuir 1999, 15, 600.
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Figure 3. Bulk vs surface concentration of UMB-C18 under the presence of the DLPC monolayer at the air/water interface: (O) Π ) 20 mN‚m-1; (b) Π ) 25 mN‚m-1.
Figure 4. A typical fluorescence intensity change during the hydrolysis reaction: (b) [UMB-C18] ) 100 nM with lipase; (2) [UMB-C18] ) 100 nM without lipase. The left-hand ordinate is expressed as the product concentration, UMB.
concentration of 100 nM, the partitioning of UMB-C18 to the monolayer increases to [UMB-C18]σ ) 4.46 × 10-2 molecule‚nm-2. The enhanced partitioning is attributed to the low initial packing density of the monolayer. Surface Pressure Dependence of Initial Hydrolysis Rate. We first show a raw hydrolysis curve in Figure 4, time dependence of fluorescence intensity. This assay was carried out on the monolayer at Π of 25 mN‚m-1, [UMB-C18] ) 100 nM and enzyme surface concentration, [lipase]σ ) 1.08 × 10-2 molecule‚nm-2. The filled triangles denote the control experiment which has been done under the same condition without lipase. It is clear that an increase in fluorescence intensity is from UMB, which is generated by hydrolysis of UMB-C18 catalyzed by lipase. After conversion of the fluorescence intensity to the concentration of the detectable product, UMB, the initial hydrolysis rate Vo is evaluated from the initial slope of the product-time curve. It has been widely accepted that the enzyme activity in monolayers at the air/water interfaces is strongly dependent on “its interfacial quality”.12 Although monolayer constituents can alter “interfacial quality”, it seems that the predominant variable for the enzyme activity in monolayers is the surface pressure. Hence, the surface pressure effect on the initial reaction rate of lipase catalysis is examined here. The DLPC monolayers were prepared at the initial surface pressure Πini of 5 mN‚m-1, and then lipase solution was injected into the subphase. The surface pressure, after allowing it to reach equilibrium partitioning of lipase to the monolayer Πeq, was 25.4 ( 0.5 mN‚m-1 for Πini ) 5 mN‚m-1. At this stage, the entire experimental protocol was repeated.
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Figure 5. Surface pressure dependence of initial hydrolysis rate as a function of bulk substrate concentration: (9) [UMBC18] ) 50 nM; (2) [UMB-C18] ) 75 nM; (b) [UMB-C18] ) 100 nM. The dashed line denotes the lipase concentration in the monolayer.
Figure 5 shows the surface pressure dependence of the initial hydrolysis rate Vo as a function of substrate bulk concentration, as well as the lipase surface concentration, [lipase]σ, denoted by the broken line. Each reaction was carried out under the condition of a constant surface pressure by moving a Teflon barrier if it was necessary. The results plotted in Figure 5 make it clear that the hydrolysis rate is maximized around Π ) 25 mN‚m-1, regardless of the substrate bulk concentration in a range of 50-100 nM. This optimal surface pressure with respect to the enzyme activity has been reported in previous pioneering works.24-27 At the Π region higher than 30 mN‚m-1, the surface pressure could not be maintained at a constant value even though the Teflon barrier was moved. This is because a fairly large amount of lipase has been continuously desorbing from the monolayer. Hence, we truncate to examine Vo at Π < 30 mN‚m-1. We revisit why a “bell-shaped” curve is manifested on the surface pressure vs hydrolysis rate relation. When the conditions of different surface pressures are attained, at least four factors which may affect the hydrolysis rate are subsequently varied. Those variables are substrate and enzyme surface concentrations, in-plane mobility of reactants, and local enzyme conformation. Two points should be made for Vo profiles in Figure 5 before we consider the four factors one by one. First, the bell shape Vo profiles rise monotonically until they reach maxima at or in the vicinity of Πeq for all three substrate concentrations. Second, Vo decreases monotonically with Π > Πeq. Considering the four factors in sequence, we can eliminate the first three but not the fourth. (1) Substrate concentration in the monolayer [UMB-C18]σ: It decreases with Π at a constant substrate bulk concentration in the subphase while Vo increases with Π e Πeq; therefore it is eliminated. (2) Lipase concentration in the monolayer [lipase]σ: It is kept approximately constant at 0.97-1.08 molecules × 100 nm-2 although it increases monotonically with Π within this narrow Π range, as indicated by the broken line with the right-side ordinate in Figure 5. Given the observed rate enhancement by a factor of 2 at Π e Πeq, it is difficult to attribute the rate change to such a minute increase in [lipase]σ; hence we eliminate this as the cause (24) Shah, D. O.; Schulman, J. H. J. Colloid Interface Sci. 1967, 25, 107. (25) Esposito, S.; Se´me´riva, M.; Desnuelle, P. Biochim. Biophys. Acta 1973, 302, 293. (26) Verger, R.; Rietch, J.; van Dam-Mieras, M. C. E.; de Haas, G. H. J. Biol. Chem. 1976, 251, 3128. (27) Pattus, F.; Slotboom, A. J.; de Haas, G. H. Biochemistry 1979, 18, 2691.
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Figure 6. Schematic representation of a possible kinetic model for the enzymatic hydrolysis reaction at the interface. Symbols follow the standard nomenclature.
of Vo at Π e Πeq. (3) In-plane mobility of the reactants: The lateral diffusion coefficients of a probe lipid in the DLPC monolayers is found to decrease with increasing Π,23 and this is consistent with increasing the in-plane viscosity of the monolayer,29 while this is contrary to the observed Vo increase with Π. Hence, we can eliminate this factor as well. (4) Lipase conformation: It has been suggested some time ago that lipase is inactivated by insufficient room for conformational variation at higher Π and by denaturation at lower Π.25 Though we cannot lend any direct support to this suggestion, we have no means to eliminate this factor unlike the previous three. Although it is almost impossible to analyze experimentally the precise lipase conformation at the air/water interface for the moment, it is most likely that the change of lipase conformation with the surface pressure is the main reason Vo increases with increasing Π in the Π range smaller than Πeq. A more detailed argument about this is necessary with the kinetic constants of interfacial hydrolysis reaction and, thus, is deferred to a later section. On the other hand, for the Π range >Πeq, all of four factors may cooperatively render Vo to decrease with Π. Kinetic Model of Enzyme Catalysis on Monolayers. We now turn to the kinetic model of the enzymatic hydrolysis reaction on monolayers at the air/water interface. Since enzyme and substrate are present in the same plane, the classical Michaelis-Menten model can be applied for our experiment after taking into account the fact that a reaction takes place only at the interface. Our kinetic model of interfacial Michaelis-Menten mechanism12-13,15-16 is represented in Figure 6. In this figure, the species marked with an asterisk designate those on the monolayer. Some fraction of the total substrate added to the subphase rapidly penetrates to the monolayer to establish the equilibrium partitioning between substrate in the subphase, S, and that in the monolayer, S*. The enzyme at the interface E* binds S* to form an (ES)* complex. Although it is actually possible that E* binds S, this elemental step might be negligible at [UMB-C18] e 150 nM. The effect of (E*S) complex formation on the hydrolysis rate will be discussed in detail in the accompanying article.30 Following its decomposition, the hydrolysis products P*, generated in the interface and are no longer surface active, desorb promptly from the interface. From the steady-state approximation, the mass conservation and the assumption that k3 is very large, the interfacial hydrolysis rate Vo can be derived as follows
Vo )
k2[E*]o[S]o k2 1 A k2 1 A + + + + [E*]o + [S]o k1 K1 V Ks V ks
(
)(
) (
)
(1)
where K1 and Ks are k1/k-1 and ks/k-s, respectively, and A and V are the total monolayer area and the volume of the system. [E*]o and [S]o are the enzyme concentration (28) Kim, S.; Yu, H. J. Phys. Chem. 1992, 96, 4034. (29) Hughes, B. D.; Pailthorpe, B. A.; White, L. R. J. Fluid Mech. 1981, 110, 349. (30) Tanaka, K.; Mecca, P. S.; Yu, H. Langmuir 2000, 16, 2672.
Figure 7. Substrate concentration dependence of initial hydrolysis rate as a function of surface pressure: (O) Π ) 20 mN‚m-1; (b) Π ) 25 mN‚m-1. The inset shows the LineweaverBurk plot; the symbols are the same as in the main plot.
in the monolayer and the total concentration of substrate in the system. We now try to establish the validity of our assumption that the adsorption of the substrate and the desorption of the products are much faster than the reaction kinetics. In the case of lipase catalysis on monolayers at Π ) 20 and 25 mN‚m-1, the moving of the Teflon barrier was unnecessary to keep a given surface pressure for at least the first 100 s, which is the necessary time scale to evaluate Vo, independent of the surface concentrations of substrate and lipase. This clearly indicates that in this Π range, the adsorption and desorption are so fast that the barostatic barrier movement is undetectable, validating our assumption. Assuming for the moment that the substrate hydrolysis is diffusion-controlled, we consider the time required for an adsorbed substrate molecule to diffuse to an enzyme. If the time is much faster than our experimental time scale of 100 s, then the stationary position of the barrier under the barostatic condition is entirely to be expected and consistent with the fast adsorption/ desorption hypothesis. We estimate the diffusion time as follows. Under our experimental conditions of [UMB-C18] ) 100 nM and [lipase]σ ) 1.08 × 10-2 molecule‚nm-2, there should be on the average one substrate and enzyme each per approximately 100 nm2 of the monolayer. The furthest distance to encounter for each other is 14.1 nm. The mean square displacement 〈r2〉 on two-dimension is 4D(2)t where D(2) and t are the lateral diffusion coefficient of diffusant and the time, respectively.31 The D(2) of a probe lipid in the DLPC monolayer at Π ) 25 mN‚m-1 is 4.1 × 10-7 cm2‚s-1.23 Assuming that the D(2) of UMB-C18 is comparable to this number and that of the enzyme is small enough to ignore, the possible longest time to encounter each other at the initial stage of reaction is calculated to be 1.2 × 10-6 s, which is much faster than the time scale of our experiment. If the adsorption of the substrate and the desorption of the products are slower than this step, a constant surface pressure cannot be attained without moving the Teflon barrier due to the accumulation of products at the surface. Kinetic Rate Constants on Monolayers. Having covered the surface concentration of substrate and surface pressure dependence of the hydrolysis rate and presented the kinetic model, we now turn to the kinetic rate constants. Figure 7 shows the substrate concentration vs the initial hydrolysis rate at two different surface pressures. Measurements were carried out under the conditions of constant temperature of 296.2 K and [lipase]σ ) (31) Crank, J. The Mathematics of Diffusion; Clarendon Press: Oxford, 1967; p 1.
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Figure 8. Enzyme concentration dependence of initial hydrolysis rate as a function of surface pressure and substrate concentration: (O) Π ) 20 mN‚m-1, [UMB-C18] ) 100 nM; (2) Π ) 25 mN‚m-1, [UMB-C18] ) 75 nM; (b) Π ) 25 mN‚m-1, [UMB-C18] ) 100 nM. The inset shows the double-reciprocal plot of [lipase]σ and Vo; the symbols are the same as in the main plot. Table 1. Rate Constants of Lipase-Catalyzed Hydrolysis Reaction on Monolayers at the Air/Water Interface Π/mN‚m-1 k1/(molec‚nm-2)-1‚s-1 k2/s-1 103ks/m‚s-1 k-s/s-1 20 25 a
16 53a 62b
33 166
1.5 1.0
2 3
[UMB-C18] ) 100 nM. b [UMB-C18] ) 75 nM.
1.08 × 10-2 molecule‚nm-2. The initial hydrolysis rate increased with the increasing substrate concentration, obeying Michaelis-Menten type kinetics. The turnover number k2 can be directly deduced from a LineweaverBurk plot which is the double-reciprocal plot of [UMBC18] and Vo as shown in the inset of Figure 7. The values of k2 so obtained at Π ) 20 and 25 mN‚m-1 are 33 and 166 s-1, respectively. The solid lines in Figure 7 are drawn according to eq 1 with the constants obtained from the Lineweaver-Burk plot. We truncate the examination of Vo at about 150 nM since Vo clearly deviates from the kinetic model shown in Figure 6, presumably because of different elemental steps.30 According to eq 1, other rate constants can be extracted from the double-reciprocal plot of [lipase]σ and Vo. Figure 8 shows the enzyme surface concentration dependence of Vo at two different surface pressures. The temperature was kept at 296 K during the hydrolysis reaction. The initial hydrolysis rate increased with the increasing lipase concentration. All of the k1, ks, and k-s values deduced from the double-inverse plot of [lipase]σ vs Vo, as shown in the inset of Figure 8, as well as k2 are collected in Table 1. An assumption of k2 , k-1, diffusion-controlled,30 was made as k1 is extracted. Thus, it was not possible to estimate k-1 from our experiment. An uncertainty of rate constants obtained here must be reported for the sake of completeness. The intercept and its standard deviation of the Lineweaver-Burk plot at Π ) 20 mN‚m-1 were 9.05 × 10-2 and 0.44 nM-1‚s, respectively. That is, the value range of the intercept is -0.350 to 0.531, resulting in the huge k2 range of 6 s-1 to ∞, although the correlation coefficient is 0.9917. On the other hand, that range for Π ) 25 mN‚m-1 is 7 s-1 to ∞. Thus, it should be stated that our result is extremely sensitive to each data point, while it has the possibility to mislead us to exaggerated or underestimated values. In any event, the k2 values obtained here are to be contrasted to values obtained in bulk solution reaction
with the same substrate as 0.10 ( 0.03 s-1.32 It should be noted that the lipase on the monolayer at the air/water interface is better activated by 330-fold at Π ) 20 mN‚m-1 and 1660-fold at Π ) 25 mN‚m-1. The interfacial activation of lipase is now reasonably well delineated to be the conformational change attending its translocation at the lipid/water interface; the lid (or flap) domain of the enzyme opens at the interface to make the active site, consisting of serine, histidine, and aspartate or glutamate, called the catalytic triad, available to substrate access.33-35 The rate constants of the catalytic step, that is k2, depended on the surface pressure, and the factor of these values between Π ) 20 and 25 mN‚m-1 was 5. This factor can be explained only in terms of local conformational change of enzyme, which might be the slight change of the relative position of the catalytic triad in the active site of lipase.36 It can be envisaged that the strong surface pressure dependence of enzyme activity observed in Figures 5, 7, and 8 is due to this local conformational change of lipase. The value of k1 at Π ) 20 mN‚m-1 was 16 (molecules‚nm-2)-1‚s-1 and was smaller than that at Π ) 25 mN‚m-1, 53-62 (molecules‚nm-2)-1‚s-1, by about a factor of 1/4-1/3. The k1 value physically means both the encounter frequency of reactants and the possibility of the successive bimolecular association after encountering. These two act on the magnitude of k1 with competition for each other as the surface pressure increases. The encounter frequency is proportional to the diffusion rate of components, substrate and enzyme, which decreases with the increasing Π. The factor of the diffusion coefficient of a probe molecule between Π ) 20 and 25 mN‚m-1 is ca. 1.2.23 On the other hand, the binding possibility of substrate to the active site of enzyme to form the transition state is strongly affected by the local conformation at the vicinity of the catalytic triad and is increased with the surface pressure until Πeq. Since the k2 might directly reflect the order of the optimization for the relative position of the catalytic triad, it can be surmised that the factor of the bimolecular association between Π ) 20 and 25 mN‚m-1 is about the same as that of k2. Thus, the discrepancy of k1 values between Π ) 20 and 25 mN‚m-1 (a factor of 1/4-1/3) can be qualitatively explained by invoking these counterbalancing factors (1.2/5 ) 1/4). More importantly, we find that the rate constant for the bimolecular association, k1, is independent of the surface concentration of reactants within the experimental error, as expressed in eq 1. The rate constants related to adsorption and desorption of the substrate, ks and k-s at Π ) 20 mN‚m-1 are larger and smaller than those at Π ) 25 mN‚m-1, respectively. This result is reasonable by the fact that the DLPC monolayer at Π ) 20 mN‚m-1 can contain the larger amount of the substrate in comparison with that at Π ) 25 mN‚m-1 due to its lower surface mass density. Conclusions Using the monolayer technique long pioneered by de Haas and Verger, we have examined kinetics of the (32) The k2 in bulk solution was measured by using a fluorescent cell with volume of 3.4 mL as a reactor. The substrate concentration region examined was 50-300 nM because the aggregation of substrate could be seen by naked eyes at a concentration higher than 400 nM. The total lipase concentration was 32 nM. Great attention was paid to avoid air bubbles in the reactor during the hydrolysis reaction. (33) Brzozowski, A.; Derewenda, U.; Derewenda, Z. S.; Dodson, G. G.; Lawson, D. M.; Turkenburg, J. P.; Bjorkling, F.; Huge-Jensen, B.; Patkar, S. A.; Thim, L. Nature 1991, 351, 491. (34) Lawson, D. M.; Brzozowski, A. M.; Dodson, G. G. Curr. Biol. 1992, 2, 473. (35) van Tilbeurgh, H.; Egloff, M.-P.; Martinez, C.; Rugani, N.; Verger, R.; Cambillau, C. Nature 1993, 362, 814. (36) Mathews, C. K.; van Holde, K. E. Biochemistry; Benjamin/ Cummings Publishing: Menlo Park, CA, 1996; p 360.
Lipase Catalysis on Monolayers
interfacial enzyme catalyzed hydrolysis reaction. The newly synthesized amphiphilic molecule, UMB-C18, was chosen as a substrate for lipase-catalyzed hydrolysis on monolayers at the air/water interface. A main requirement for the substrate is fast adsorption to monolayers and fast desorption of the products into bulk phase after hydrolysis. The optimum surface pressure related to the initial hydrolysis rate was observed at around Π ) 25 mN‚m-1, which is the equilibrium surface pressure in terms of the lipase adsorption. The turnover numbers, k2, on monolayers at Π ) 20 and 25 mN‚m-1 are 33 and 166 s-1, respectively, and are much larger than that in bulk phase of 0.1 s-1. The interfacial activation of lipase is interpreted in terms of the translocation of a lid domain of the enzyme to open at the interface to allow the active site available to substrate approach. The surface pressure dependence
Langmuir, Vol. 16, No. 6, 2000 2671
of the turnover number is speculated by invoking the change of the relative position of the catalytic triad of lipase. Finally, it was shown on the basis of k1 that the binding of substrate to the active site of enzyme strongly depends on the surface pressure by virtue of the local conformational change as well. Acknowledgment. This work was partially supported by the Eastman Kodak Professorship and NSF grants (DMR9203289 and DMR9711226) awarded to H.Y. and a Biophysics Training Grant of NIH (No. 5T32 GM08293) awarded to P.A.M. We are most grateful for helpful discussions with Professor George Zografi, and for the substrate synthesis by Dr. David D. Manning. LA9909244
