Kinetics of Detergent-Induced Activation and Inhibition of a Minimal

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Kinetics of Detergent Induced Activation and Inhibition of a Minimal Lipase Daniel Kuebler, Anna Bergmann, Lukas Weger, Kim Nadine Ingenbosch, and Kerstin Hoffmann-Jacobsen J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/acs.jpcb.6b11037 • Publication Date (Web): 20 Jan 2017 Downloaded from http://pubs.acs.org on January 27, 2017

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The Journal of Physical Chemistry

Kinetics of Detergent Induced Activation and Inhibition of a Minimal Lipase Daniel Kübler, Anna Bergmann, Lukas Weger, Kim N. Ingenbosch, and Kerstin HoffmannJacobsen* Niederrhein University of Applied Sciences, Department of Chemistry, Adlerstr. 32, 47798 Krefeld, Germany

ABSTRACT

Detergents are commonly applied in lipase assays to solubilize the sparingly soluble model substrates. However, detergents affect lipases as well as substrates in multiple ways. The effect of detergents on lipase activity is commonly attributed to conformational changes in the lid region. This study deals with the effect of the non-ionic detergent, polyethylene glycol dodecyl ether, on a lipase that does not contain a lid sequence, lipase A from Bacillus subtilis (BSLA). We show that BSLA activity depends strongly on detergent concentration and the dependency profile changes with pH. The interaction of BSLA with detergent monomers and micelles is studied with fluorescence correlation spectroscopy, time-resolved anisotropy decay and temperature induced unfolding. Detergent-dependent hydrolysis kinetics of two different substrates and two pH values are fitted with a microkinetic model. This analysis shows that the 1

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mechanism of interfacial lipase catalysis is strongly affected by the detergent. It reveals an activation mechanism by monomeric detergent that does not result from structural changes of the lipase. Instead we propose that interfacial diffusion of the lipase is enhanced by detergent binding. Introduction Lipases (EC 3.1.1.3) catalyze the hydrolysis of triglycerides. These substrates are virtually insoluble in water 1. In contrast to esterases, lipases have the characteristic property to show a higher activity towards the aggregated state 2. Lipases specifically perform interfacial catalysis. In order to analyze lipase activity by spectroscopic assays, model substrates have to be used. Typically, the hydrolysis is monitored with the use of chromogenic or fluorogenic esters, e.g. pnitrophenol esters. Detergents are frequently applied in lipase assays in order to solubilize and control the dispersity of the sparingly soluble substrates 3. The substrates are included in micelles forming a biomimetic lipid surface. It is known that detergents influence lipases, as well as substrates, in various ways 4. First, the substrate microenvironment is varied from substrate aggregates to a micellar system. Second, detergents can interact with lipases and alter their structure and activity. Ionic surfactants prevalently affect protein conformational stability 5. Neutral detergents have a less pronounced effect on protein structure. Yet, neutral detergents can lead to lipase inhibition, activation or lipase superactivity. Lipase inhibition has been rationalized by solubilizing the lipase as well as the substrate, thus lowering the mutual affinity 6 or blockage of the active side 7. Most lipases have a “lid domain” protecting the hydrophobic active center which opens upon interaction with 2


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the oil/water interface. Crystallographic analysis of lipases bound to detergents reveal that detergent monomers preferentially bind to the region of the active site of lipases 8. Detergent binding has been observed to change the lid conformation to the open state 9,10, which is associated with an increased activity. Superactivity has been attributed to an extended stabilization of the “open” conformation in a highly hydrophobic environment provided by micellar systems 11. A strict mathematical description of interfacial enzyme kinetics is hampered by the difficulty to obtain the relevant experimental data to verify a model. In such a model the partitioning of enzyme, as well as substrate in the aqueous and lipidophilic phase has to be quantified. It has been suggested that the macroscopic hydrolysis kinetics of lipases can be described by the sum of the kinetics of all reactions between substrate and lipase species present, if the partition equilibria are known. A number of kinetic models have been proposed 12,13. However, due to the complexity and the amount of species involved, the models could not be unambiguously approved on the basis of experimental data 14,15.

Bacillus subtilis lipase A, BSLA, is the smallest lipase known to date. It shows a minimal α/βhydrolase fold but is considered to lack a lid covering the active site 16,17. However, it is classified a lipase as it preferentially hydrolyzes long chain triglycerides 18,19. In the present study we use BSLA as a minimal lipase model to analyze the influence of non-ionic detergents on lipase catalysis excluding lid properties. BSLA has been shown to be influenced by the presence of detergents in an activating or inhibiting manner 20,21. Thesit® is used as a nonfluorescent model detergent for polyethylene glycol ether detergents as TritonTM-X 100, that are 3



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commonly applied in lipase assays or lipase catalysis. We aim to derive a detailed mechanism of the deviated interfacial catalysis in the presence of non-ionic detergents. Fluorescence correlation spectroscopy (FCS) is applied to analyze BSLA micelle binding. Temperature induced unfolding allows the analysis of detergent binding. Fluorescence spectroscopy of tryptophan single mutants is used to detect detergent-induced conformational changes of BSLA. These results are fed into a microkinetic fit to the detergent dependent rate of hydrolysis. We have shown recently that the alkalophilic BSLA adopts a highly hydrophobic and active state at pH 8.5, whereas it is more stable but less reactive at pH 10 22. In this study, we juxtapose the interfacial kinetics of these two species. By global fitting the kinetic data of both species, the unambiguity of the fit is reduced. Experimental Methods Protein Purification The gene of BSLA in the pET 28(a) vector was kindly provided by Dr. Ulrich Krauß (Forschungszentrum Juelich). The wild type (WT) protein was expressed and purified as described earlier 22. The mutants W42C, W42F and W31F were prepared by the QuikChange protocol as described 22. W42F and W31F were purified by nickel affinity chromatography. W42C was purified by cation exchange chromatography in the presence of 2 mM dithiothreitol. Buffer composition BSLA was buffered with a 20 mM sodium phosphate buffer (pH 8.5) or 10 mM glycine buffer (pH 10).

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Enzymatic Activity 4-methylumbelliferyl (4-MU) oleate, 4-MU butyrate and Thesit (polyethylene glycol dodecyl ether) were purchased from Sigma Aldrich. Substrates were prepared as DMSO stock solutions and diluted to 6 µM in the assay. A final concentration of 2% DMSO was present in all measurements. The reaction was started by the addition of enzyme in a final concentration of 5 nM. Reaction temperature was 20°C. Kinetics were measured with a Varian Cary Eclipse Spectrofluorometer (Excitation 327 nm, Emission 449 nm) providing a time resolution of 300 ms at the beginning of the time traces. In order to convert fluorescence intensity data into concentration data, 4-methylumbelliferone was used for calibration. The initial rate was determined in the respective linear region of each time trace. Data analysis Kinetic simulations were carried out with MATLAB (Mathworks, Natick, MA). Non-linear least square regression was performed with the lsqcurvefit routine. The kinetic model bases on the work of Viparelli et al 23. Briefly, the model takes into account that enzyme as well as substrate may exist as different species. Molecular substrate and substrate associated with micelles are in equilibrium (KS). The enzyme can exist as free enzyme as well as in micelle-bound (Kmic) form. Additionally, we consider the equilibrium of the free enzyme with enzyme bound to monomeric detergent (Kmon). Viparellis model assumes Michaelis-Menten kinetics for all reactions of the permuted reaction partners. We use the turnover rate as quantitative measure for the reactivity of the different species. The initial rate is calculated based on the equilibrium and kinetic parameters and compared to the experimental rate. 5



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Fluorescence spectroscopy Protein steady-state fluorescence emission was analyzed with a Varian Cary Eclipse fluorometer. Temperature-induced unfolding was performed with a Peltier temperature control, excitation wavelengths of 280 and 295 nm and an emission wavelength of 335 and 358 nm. The slit widths of excitation and emission monochromators were set to 5 nm. Protein concentration was 5 µM for pH 10 und 2 µM for pH 8.5. Unfolding transitions obtained at both excitation wavelength were analyzed as described by Santoro and Bolen 24, assuming a two-state unfolding model. The free enthalpy of unfolding, ∆H°, was determined by a global fit of the emissions at 335 und 358 nm at all excitation wavelengths to Eq. 1.

=

Δ° − + ∙ + + ∙ ∙ exp ∙

Δ° − 1 + exp ∙

(Eq. 1)

y depicts the measured fluorescence intensity. yu and yf are the fluorescence intensities of the folded and unfolded protein, mf and mu are the slopes of the pre- and post-transition lines. Tm is the melting temperature midpoint. Fluorescence anisotropy Fluorescence anisotropy was measured by time-resolved fluorescence spectroscopy. Measurements were carried out with a FluoTime 200 instrument from Picoquant (Berlin) equipped with Glan-Taylor polarizers. A PLS 290 laser diode (Picoquant, Berlin) was used for 6


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excitation (excitation wavelength: 291 nm +/- 9.6 nm) and a UV short pass filter (250-340 nm). Emission wavelength was 335 nm, slit width 2 nm. The sample temperature was 20°C. Anisotropies r were fitted to a sum of exponentials model 25 #

= +

"

(Eq. 2)

!

%$where is the residual anisotropy and & are the respective correlation times. The FluoFit software provided by the instrument manufacturer was applied using the tail fit mode. &% was fixed to 8.45 ns to account for global protein tumbling as determined earlier 22. Fluorescence correlation spectroscopy FCS experiments were carried out using a home-built confocal microscope. The system is equipped with an oil immersion alpha Plan-Apochromat 63x/1.46 Oil Corr M27 / NA 1.46 (Zeiss, Jena, Germany) objective. The collar setting of the objective was set to 0.17. The sample was illuminated by a 482 nM diode laser (LDH P-C-485, Picoquant, Berlin, Germany), focused through the objective, at a power level of 2 mW. Fluorescence was collected through the objective, and was then filtered by a dichroic mirror (H 488 LPXR superflat Vers.2, AHF Analysentechnik, Tübingen, Germany) and a fluorescence filter (10XM20-485, Newport Corporation, Irvine, CA). The fluorescence was then focused by the microscope tube lens onto a 50 µm pinhole to filter out-of-focus light. It was split by a nonpolarizing beam splitter and focused on a camera (Lumenera Infinity3-1UR, Ottawa, ON) and a single-photon counting hybrid photodiode (PMA Hybrid-06, Picoquant, Berlin, Germany). Data collection and 7



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generation of FCS curves were carried out using a hardware correlator (PicoHarp 300, Picoquant, Berlin, Germany). Experimental correlation functions were fit to the equation appropriate for a 3D Gaussian beam shape: ( % ( %⁄. ' ( = ') ∙ *1 − , ∙ *1 − . , + - +

(Eq. 3)

where G0 is the reciprocal of the total average number of molecules in the observation volume, τ is the mean diffusion time of a molecule through the observation volume, and κ is the ratio of the . squared beam waists (given by - = 01. "023 ). The diffusion coefficient is related to the

parameters above through the relation 4 = 023 ⁄4+ . The data was fitted to a model of two diffusion times, one accounting for residual free fluorescence label and the second describing the labeled lipase. The introduction of a third diffusion time to differentiate bound and free lipase decreased the quality of the fit results and the stability of the fit significantly. The mutant W42C was labeled with Atto-488 maleimide (Atto-tec, Siegen, Germany) according to the manufacturer´s protocol. The final protein concentrations were typically 30–40 nM. Microscope covers slips were pegylated with 3-[Methoxy(polyethyleneoxy)propyl]trimethoxysilane (ABCR Chemie, Karlsruhe, Germany). The set-up was calibrated with Atto-488 NHS ester (Atto-tec, Siegen, Germany) as described 26.

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Results Lipase detergent interactions Fluorescence correlation spectroscopy (FCS) measures the fluctuations of fluorescence emitted from molecules diffusing through a small illumination volume of a laser beam focused by a microscope objective 27,28. It detects the translational diffusion times of molecules which is related to their diffusion coefficient D. The radius of hydration RH can be calculated from the diffusion coefficient with the Stokes-Einstein relation, if the viscosity η is known

4=

67 69:;

(Eq. 4)

where kB is Boltzmann´s constant and T is the temperature. FCS has been effectively applied to study the radius of hydration of proteins 29. We use FCS to investigate the interaction between BSLA and detergent micelles. BSLA bound to detergent micelles is expected to have an increased radius of hydration than free BSLA. Hence, FCS can be used to detect binding events between BSLA and micelles. Figure 1 shows the autocorrelation curves of fluorescence-labeled BSLA in the absence and the presence of increasing Thesit concentration fitted to Eq. 3 at pH 8.5 and 10. The first detergent concentration (233 µM) is well beyond the cmc (100 µM) and the second (350 µM) represents an increased micelle concentration. On the left panel, BSLA binding analysis at pH 8.5 is shown. The presence of Thesit micelles leads to an increased diffusion time which reflects an increased radius of

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hydration. When the Thesit concentration is increased to 350 µM, RH increases further (Table 1).

Figure 1. Fluorescence correlation curves of W42C-Atto 488 at pH 8.5 (left) and pH 10 (right). The colors display the detergent concentration. Black: no detergent, red: 233 µM Thesit, blue: 350 µM Thesit. All curves were fitted to Eq.3. Weighted residuals as provided by the SymphoTime software of the fit of the pure lipase are given exemplarily at the bottom of the figure. At pH 10 the autocorrelation curves remain unchanged upon the addition of Thesit micelles. The variations in the ms-regime result of different abundance of triplet states. Hence, FCS analysis reveals that BSLA is capable of attaching to Thesit micelles at pH 8.5 whereas no BSLA micelle interaction takes place at pH 10. The experimentally determined radius of hydration of BSLA of 2.0 ± 0.2 nm can be compared to a computational prediction deduced from crystal structure data with the algorithm developed by 10


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Garcia de la Torre et al 30. Using the program HYDROPRO and the PDB data 1ISP 31, we obtained a theoretical RH of 2.0 nm. This is in excellent agreement with our measured value. From literature data we estimate the radius of polyethylene glycol dodecyl ether micelles to 1.5 nm 32–35. Thus, BSLA micelle aggregates must exceed a RH of 3.5 nm. The RH of 4.4 ± 1 nm determined at 233 µM is in good agreement with this expectation. It must be noted that we determined only one mean diffusion time of the lipase. Thus, the large error bar supposedly includes the distribution of species (free and bound lipase). At high micelle concentrations larger micelle-lipase aggregates are found which lead to visible precipitation at prolonged incubation times. Table 1: Detergent dependence of hydrodynamic radii of BSLA at pH 8.5 and pH 10 determined by FCS.

[Thesit]

RH (pH 8.5)

RH (pH 10)

0

2.0 ± 0.2 nm

1.9 ± 0.2 nm

233 µM

4.4 ± 1.0 nm

2.0 ± 0.2 nm

350 µM

9.2 ± 5.0 nm

1.7 ± 0.2 nm

Thermal unfolding of BSLA in the presence of detergent was investigated in order to study the thermodynamics of BSLA detergent interaction. Unfolding was analyzed by tryptophan fluorescence intensity at two excitation (280,295 nm) and two emission (335, 358 nm)

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wavelengths. The global fit to Eq. 1 of the four denaturation curves was used to determine the enthalpy of unfolding (∆H°unf) as a function of detergent concentration (Figure 2, Figure S3).

Figure 2. Van’t Hoff enthalpy of protein unfolding ∆H°unf versus Thesit concentration (pH 8.5 (red), pH 10 (black)). Error bars reflect the error of repetitive measurements. The enthalpy of unfolding can be converted to the free energy of unfolding using the melting temperature Tm and the heat capacity of unfolding ∆Cp,unf 36. We refrained from performing this calculation and discuss enthalpies. First, the melting temperature is another error-prone fitting parameter. Second, ∆Cp,unf is usually estimated and the influence of detergent on ∆Cp,unf is not known. In the absence of detergent BSLA shows a higher enthalpy of unfolding at pH 10 than at pH 8.5. In the sub-cmc region, the enthalpy of unfolding decreases linearly with increasing detergent concentration. This effect is more pronounced in the case of the isoform of higher ∆H°unf at pH 10. This is a strong indication that BSLA is capable of interacting with monomeric detergent at 12


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both pH, which is observed as exothermic binding. It should be noted, that the changes in ∆H°unf with detergent concentration cannot be considered exact binding energies as the reference state is the unfolded lipase in detergent solution and the unfolded state can include lipase detergent binding, too. However, exothermic binding of detergents is usually found for specific highaffinity binding 5, which supports the idea of specific binding of monomeric detergent to BSLA. Above the cmc, enthalpy increases with increasing detergent concentration at pH 8.5. At pH 10 ∆Hunf does not show a similar trend. This agrees with the observation made with FCS that only the hydrophobic state of BSLA at pH 8.5 is capable of attaching to detergent micelles. BSLA micelle binding is observed as endothermic binding in the thermal unfolding experiment. This is in line with a calorimetric analysis of the interaction of the non-ionic detergent Triton X-100 with Bovine Serum Albumin 37. Here, endothermic protein micelle binding was found. Yet, the enthalpy difference of BSLA bound to detergent to free BSLA may account in parts for structural changes of the protein in the presence of detergent. In order to study potential conformational changes of BSLA upon interaction with detergent, fluorescence depolarization was applied. Fluorescence anisotropy decays provide information on the diffusive motion of a fluorophore during its exited state 38,39. BSLA has two native tryptophan residues, W42 and W31. In a previous study we have shown, that the local motion of the respective single tryptophan residues can be analyzed by a two-exponential decay model 22 to detect structural variations of BSLA. The rotational correlation time of &% = 8.45 ns accounts for global protein tumbling. A second, faster component (&. ) has been observed that arises from restricted local motion of the 13



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tryptophan side chain and is a sensitive probe for the local protein structure. The anisotropy decay W31F and W42F was measured in increasing amounts of Thesit from buffer to cmc.

Figure 3. Fluorescence depolarization of W31 F at pH 8.5 (A) and pH 10 (B) in the presence of 100 µM Thesit (red) and in buffer (green). The lines represent fits to Eq. 2. Bottom: Weighted residuals of the fit (100 µM Thesit) as provided by the FluoTime software. Figure 3 depicts the anisotropy decay traces of W31F in Thesit solution (cmc) and in the absence of detergent. This mutant is especially interesting as W42 is probed, which is close to the active site. The overall anisotropy of W31F and W42F (data not shown) remain unchanged at pH 10 upon the addition of increasing amounts of detergent. At pH 8.5 slight variations of the anisotropy decay traces are observed. These definitely cannot be described with a change of &. indicative of local structure. The anisotropy can be rather described by an increase of &% . Thus, these changes are supposed to account for lipase micelle interaction. A rigorous quantification is not possible. However, it can be omitted without loss of insight as lipase micelle interactions have been analyzed accurately with FCS. 14


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More importantly, fluorescence anisotropy data give strong evidence that the interaction of monomeric detergent with lipase proceeds without structural changes of the lipase – especially not in the region of the substrate cleft. This conclusion includes the interaction of BSLA with micellar detergent at pH 8.5 at the cmc. Enzyme Kinetics Enzyme activity was assessed by the hydrolysis of long chain (oleate) and short chain (butyrate) carboxyl esters releasing the fluorescent alcohol 4-MU. Figure 4 plots the initial rate of 4-MU butyrate hydrolysis versus detergent concentration. Due to the enhanced autohydrolysis of 4-MU butyrate at pH 10, this analysis could only be performed at pH 8.5. Figure 4 illustrates that the activity of BSLA at pH 8.5 decreases slowly and constantly with detergent concentration. Hence, there is no indication of detergent induced activation but a slight inactivation is observed. 4-MU oleate is less prone to autohydrolysis 22. Therefore, hydrolysis of 4-MU oleate by BSLA could be analyzed at pH 8.5 and pH 10. As shown in Figure 5, the rate of oleate hydrolysis by BSLA shows a different detergent dependency than butyrate hydrolysis. Here, activity maxima are observed. Moreover, the detergent dependency of activity varies significantly with the two pH values under investigation. As illustrated in Figure 5A, lipase activity is first reduced in the regime of low detergent concentrations at pH 8.5. An activity maximum is found slightly below the cmc. BSLA stays significantly active well above the cmc. At pH 10, the activity maximum is observed at about half the cmc and activity is strongly reduced beyond the cmc (Figure 5B). Analogical kinetics were found for the detergent Triton X-100 (see Figures S1 and S2, Supporting Information). The activity maxima coincide, if the detergent concentrations are given 15



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in relation to the cmc. Triton X-100 is also a polyethylenoxide surfactant but it has an aromatic hydrophobic group. In order to deduce the mechanism of the influence of polyethylene glycol dodecyl ether on BSLA catalysis, the kinetic data was fitted to the refined kinetic model of Viparelli et al 23. In this model the interaction of the lipase with detergent micelles is expressed as partition equilibrium (Kmic). The incorporation of substrate in micelles is treated accordingly (KS). The resulting enzyme and substrate species are assumed to react subsequently with Michaelis-Menten type kinetics. As our experimental data emphasize the relevance of monomeric detergent, an additional equilibrium involving free lipase and monomeric detergent was introduced (Kmon). This model is sketched in Scheme 1. Fitting parameters were the kinetic Michaelis-Menten parameters of all possible reactions, the equilibrium constants of the detergent protein and detergent substrate species. Yet, we refrained from relying on Michaelis-Menten behavior in data analysis. We specify the turnover rate calculated of the Michaelis-Menten constants as quantitative measure for the activity of the different species.

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Scheme 1. Kinetic model for interfacial catalysis in the presence of detergent. K denotes partition equilibria and TO turnover rates of the indicated species. The first index of turnover rates labels the enzyme species and the second the substrate species (f: free, mon: bound to monomeric detergent, mic: bound to detergent micelles, b: substrate bound in micelles).

To reduce the unambiguity of the fit, equilibrium parameters were tackled first. 4-MU butyrate is not suspected to incorporate significantly into micelles. This is corroborated by the constant initial rate of 4-MU butyrate hydrolysis at detergent concentrations beyond the cmc (Figure 4). Hence, the micelle substrate partition can be neglected in the fit, reducing the fitting parameters to Kmon, Kmic and the turnover rates of the reactions with the free substrate. Kmon and Kmic are enzyme parameters so that these are global parameters for both substrates. Kmon and Kmic obtained by a fit to 4-MU butyrate hydrolysis kinetics were used as starting values for 4-MU oleate kinetics simulations at pH 8.5. These fit results were again fed back to butyrate kinetics. The procedure was continued until the fitting results converged. KS is a global parameter for all kinetic data obtained with 4-MU oleate. Thus, a second consistent fitting procedure was 17



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performed with the kinetic data given in Figure 5 to determine KS. The established equilibrium constants were locked. In the final fit the kinetic variables were optimized. KS was locked to the value at pH 8.5 in the fit to the data obtained at pH 10. As fluorescence depolarization did not show any evidence of BSLA micelle binding Kmic was estimated to zero at pH 10. It must be noted, that Kmic might well have a small but non-zero value. However, this evidence-based assumption reduces significantly the correlation of fit parameters so that the overall accuracy of the fit parameters is enhanced. Fitting parameters for the detergent dependent 4-MU oleate hydrolysis rate at pH 10 were Kmon and the kinetic parameters. The results of the fits are given in Table 2 and are visualized in Figures 4 (4-MU butyrate) and 5 (4-MU oleate). The errors on the parameters calculated with the Jacobian matrix are provided in Table 2. However, the effective errors are suggested to be reduced by the iterative fitting procedure described. Even within the framework of the conservative error estimation given in Table 2 the dominating reaction paths can be resolved as a function of detergent concentration. Figure 4 shows that the slight reduction of BSLA activity measured as 4-MU butyrate hydrolysis results of a reduced turnover rate of BSLA bound to monomeric and micellar detergent. On the contrary, 4-MU oleate hydrolysis kinetics at pH 8.5 is dominated by enzyme bound to monomeric detergent reacting with substrate incorporated in detergent micelles (Figure 5A). The activity maximum results of the maximum coexistent abundancy of those most reactive species. The low binding affinity of BSLA to micelles at pH 10 found at equilibrium is echoed in the kinetics: The turnover rates for the substrate hydrolysis at the micelle interface are very low, too. This remains unchanged when BSLA binds monomeric detergent. 18


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At pH 10 binding of monomeric detergent also leads to enzyme activation. However, as BSLA hardly converts substrate incorporated in micelles at pH 10, the activity maximum is shifted to lower detergent concentrations compared to pH 8.5. Thus, the activity maximum at pH 10 results again of the maximal abundancy of the most reactive enzyme and substrate species, which are enzyme bound to monomeric detergent and free substrate. Activity approaches zero beyond the cmc, because the free substrate is diluted out into non-accessible micelles (Figure 5D).

Figure 4. A: Initial rate of 4-MU butyrate hydrolysis at pH 8.5 versus Thesit concentration. The red line shows the fit to the data. The initial rate calculated for the relevant species are in dashed lines (green: free enzyme; blue: enzyme bound to monomeric detergent, green: enzyme bound to micelles). B: Relative enzyme concentrations of the free enzyme (black), enzyme bound to monomeric detergent (blue) and enzyme bound to detergent micelles (green).

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Figure 5. A+B: Initial rate of 4-MU oleate hydrolysis at pH 8.5 (A) and pH 10 (B) versus Thesit concentration. Experimental data are black data points. The red lines show the fit to the data. The initial rates calculated for the relevant species are given as dashed lines. The dashed black line shows the rate of the free enzyme and free substrate species, the dashed blue line the rate of the enzyme bound to monomeric detergent reacting with free substrate and the dashed orange line shows the rate of the reaction between enzyme bound to monomeric detergent and micelle bound substrate. The other rates remain zero over the Thesit concentration. C+D: Relative enzyme concentrations of the free enzyme (black), enzyme bound to monomeric detergent (blue) and enzyme bound to detergent micelles (green). Substrate species are represented as dashed lines: free substrate (magenta) and substrate bound to micelles (cyan).

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Table 2: Equilibrium constants and turnover rates of the competing reactions determined by kinetic modelling (compare Scheme 1). The equilibrium constants are given in µM-1 and the turnover rates in µM-1.s-1. The errors on the parameters are calculated with the Jacobian matrix of the fit.

Kmon

Kmic

KS

TOf,f

TOmon,f

TOmic,f

TOf,b

TOmon,b

TOmic,b

Butyrate pH8.5

0.012 ±0.002

90 ±60

-

2.0 ±0.6

0.61 ±0.83

0.63 ±0.55

-

-

-

Oleate pH8.5

0.012 ±0.001

90 ±14

2.9·103 ±0.7·103

1.9 ±0.2

0.41 ±0.36

0

0

27 ±9

0

Oleate pH 10

0.013 ±5·10-4

0

2.9·103 ±0.3·103

1.7 ±0.5

21.7 ±1.1

0

0.74 ±9.9

0

0

Discussion The mechanism of detergent interaction on lipase catalysis has been challenged for decades. The analysis is complex due to the fact that the detergent will not only influence the lipase itself but also vary the microenvironment of the interface where catalysis takes place. Most studies presume that the opening of the lid upon binding of detergents is the main root cause for detergent induced activation. In this study we aim to reveal the influence of non-ionic detergents on interfacial lipase catalysis apart from conformational changes of the lid. A kinetic modelling 21



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is used to deduce the mechanism of interfacial lipase catalysis in the presence of non-ionic detergents. Thus the lid-less lipase BSLA is used as model system. Two different conformations of the lipase present at different pH are juxtaposed. At pH 8.5 BSLA is hydrophobic and attaches to Thesit micelles. The less hydrophobic and more stable conformation at pH 10 does not associate with micelles. Due to these contrasting affinities, the effect of micelle adsorption on BSLA activity can be studied. Moreover, the experimental data basis for quantitative kinetic analysis is enlarged by studying one enzyme at different pH values. The experimental constraints of the kinetic model are expanded further by parallel analysis of a soluble and an insoluble substrate. The kinetic fits show a similar affinity of BSLA at both pH to detergent monomers. This is in line with a calorimetric analysis of the interaction between Bovine Serum Albumin and non-ionic surfactants 37. It was shown that binding of monomeric detergent occurs at specific binding sites, whereas binding to micelles is a cooperative process with the protein surface. We have shown previously that the pH influences the area of hydrophobic surfaces of BSLA which maximizes at pH 8.5 22. Hence, the affinity of BSLA at pH 8.5 to micelles can be assigned to large hydrophobic patches. The results of the current study imply that docking sites for monomeric detergent are present at any pH. These sites are suggested to be located in the region of the substrate binding pocket of lipases. This idea is supported by the minor inhibitive effect of detergent on 4-MU butyrate hydrolysis. However, we cannot find any spectroscopic evidence that binding goes along with conformational changes of BSLA. Beyond fluorescence anisotropy we analyzed fluorescence intensity spectra and the fluorescence lifetime of single tryptophan mutants of BSLA (data not shown). No significant influence of detergent was observed in any of 22


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these experiments. The lack of conformational changes agrees with the missing lid and implies that BSLA is slightly inhibited by detergent sterically blocking the active site. However, a different picture is obtained when the insoluble substrate, 4-MU oleate, is used. In this case binding of monomeric detergent induces activation. The kinetic modelling reveals that monomeric detergent activates BSLA at both pH, but the preferred substrate state is different at the pH values studied. Using 4-MU oleate interfacial catalysis at the lipid/water interphase is studied. This applies for both substrate species, micelle incorporated substrate and “free” substrate. In the latter case the substrate will exist as ill-defined aggregates. We conclude, that detergent activation of lipases exists beyond interactions of detergents with the lipase lid but represents the boost of interfacial catalysis. It has to be discussed how binding of monomeric detergent can enhance interfacial catalysis such that even the effect of slight active site blocking is balanced out. It has been suggested that detergent activation is a result of reduced lipase denaturation at the interface 40. However, we could not find any evidence of BSLA stabilization by monomeric Thesit in temperature-induced unfolding analysis. The first step of interfacial catalysis is lipase adsorption at the oil water interface. In standard kinetic experiments this step cannot be resolved. An apparent turnover rate including adsorption and catalytic step is obtained instead. As major conformational changes have been excluded, the detergent is supposed to influence lipase adsorption. Detergents reduce interfacial tension. This effect should lead to a decreased affinity of lipases bound to detergents to hydrophobic interfaces. Consequently, the rate of the initial adsorption step is reduced. If the adsorption step is 23



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rate-limiting, lipase kinetics show a lag phase 41. We find lag-phase kinetics for the hydrolysis of 4-MU oleate by BSLA but only at pH 10 and beyond the cmc. Here BSLA has to convert the substrate incorporated into micelles, with which BSLA hardly interacts at pH 10. In the monomeric activation regime, no lag-phase is observed. The critical feature of the dynamics of enzyme adsorption and desorption at the water-lipid interface has been pointed out in the context of phospholipase catalysis 42. It has been suggested that phospholipases are hopping or scooting the interface to catch a substrate. The diffusion constant of the lipase from Thermomyces lanuginosus (TLL) at hydrophobic silanized interface has been shown to increase by an order of magnitude in the presence of a mixture of non-ionic and ionic surfactants 43. We suggest that binding of monomeric detergent predominantly increases the interfacial dynamics of BSLA at the hydrophobic interface and accelerates the screening process for substrate. This idea is bolstered by the finding that the specific affinity of the two BSLA forms towards micellar and free substrate species stays unchanged upon binding of detergent but the turnover rate increases. However, we cannot judge, whether this effect includes the facilitation of substrate transfer in the interface, too. Finally, we address the question from the practical point of view whether non-ionic detergents should be used in activity assays. Our kinetic analysis of BSLA activity at pH 8.5 and 10 shows that by comparing the hydrolysis rate of both species different processes are analyzed. In one case we observe interfacial catalysis at micelles and in the other at substrate aggregates. Moreover, the kinetics are dominated by lipase activated by detergent binding. A comparative lipase assay will convolute activity with the specific activation potential of each lipase. It is 24


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questionable whether this is a property relevant for catalysis under “real” reaction conditions. However, neither the use of soluble substrate nor undefined substrate aggregates account for realistic interfacial catalysis. There are attempts to build intrinsically interfacial assays as e.g. 44. It must be a matter of future studies to develop further assays that yield realistic and reliable data on interfacial catalysis without the use of co-solvents.

Conclusion We have investigated the effect of non-ionic polyethylene glycol ether detergent Thesit on a lidless lipase. The activation and deactivation with detergent concentration could be successfully modeled with a microkinetic model. The model describes interfacial partitioning of lipase and substrate in terms of thermodynamic equilibria and accounts for all possible reactions between the substrate and lipase species assuming Michaelis-Menten kinetics. By introducing monomeric detergent species into the model the experimental data could be fit. It is found that monomeric detergent is fully responsible for the interfacial activation although no conformational change of the lipase takes place. We suggest that diffusion of the lipase at the water/lipidophilic interface is enhanced. Appendix The cmc of Thesit was determined using pyrene according to Molina-Bolivar et al 45. This data was used to determine the concentrations of the detergent species (monomeric detergent,

Kinetics of Detergent-Induced Activation and Inhibition of a Minimal

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