Trehalose Mediated Inhibition of Lactate Dehydrogenase from Rabbit

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B: Biophysical Chemistry and Biomolecules

Trehalose Mediated Inhibition of Lactate Dehydrogenase from Rabbit Muscle. The Application of Kramers´ Theory in Enzyme Catalysis Juan M. Hernández-Meza, and José G. Sampedro J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/acs.jpcb.8b01656 • Publication Date (Web): 29 Mar 2018 Downloaded from http://pubs.acs.org on March 29, 2018

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The Journal of Physical Chemistry

Trehalose Mediated Inhibition of Lactate Dehydrogenase from Rabbit Muscle. The Application of Kramers´ Theory in Enzyme Catalysis Juan M. Hernández-Meza and José G. Sampedro* Instituto de Física, Universidad Autónoma de San Luis Potosí. Manuel Nava 6, Zona Universitaria, C.P. 78290. San Luis Potosí, SLP. México

1 Environment ACS Paragon Plus

The Journal of Physical Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ABSTRACT: Lactate dehydrogenase (LDH) catalyzes the reduction of pyruvate to lactate by using NADH. LDH kinetics has been proposed to be dependent on the dynamics of a loop over the active site. Kramers´ theory has been useful in the study of enzyme catalysis dependent on large structural dynamics. In this work, LDH kinetics was studied in the presence of trehalose and at different temperatures. In the absence of trehalose, temperature increase raised exponentially the LDH Vmax and revealed a sigmoid transition of Km towards a low-affinity state similar to protein unfolding. Notably, LDH Vmax diminished when in the presence of trehalose, while pyruvate affinity increased and the temperature-mediated binding site transition was hindered. The effect of trehalose on kcat was viscosity dependent as described by Kramers´ theory since Vmax correlated inversely with the viscosity of the medium. As result, activation energy (Ea) for pyruvate reduction was dramatically increased by trehalose presence. This work provides experimental evidence that the dynamics of a structural component in LDH is essential for catalysis, i.e. the closing of the loop on the active site. While the trehalose mediated-increased of pyruvate affinity is proposed to be due to the compaction and/or increase of structural order at the binding site.


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INTRODUCTION The enzyme lactate dehydrogenase (LDH, E.C. 1.1.1.27) performs the conversion of pyruvate to lactate by using NADH as reducing molecule.1 LDH is present in mammal tissues displaying highly active glycolysis such as muscles under intense exercise (anaerobic fermentation), red cells, cancer cells2,3 (aerobic glycolysis/Warburg effect), and others. LDH is a tetramer and depending on the tissue, it is formed or not by a combination of different isozymes.4 All LDHs essentially share the same structural topology and identical binding site structure.5 LDH kinetics and catalytic mechanism have been of high interest,6,7 i.e., it has been proposed that both depend on the dynamics of LDH structure8,9 as a whole and locally by the closing of a loop above the substrate binding site.7,10 Further, LDH is important as a monitor of human health status11,12 (the assay of LDH activity is routinely performed in clinical laboratories)13 and recently has come to be a target for development of inhibitors for cancer treatment.3,14–19 In addition, the use of LDH as biosensor has gained high interest and thus the study of the enzyme under nonstandard conditions is of utmost importance.20–22 LDH has been used also as a model enzyme in protein stability studies.23–27 In this regard, it is known that protein unfolding involves the diffusion of protein structure away from the native state.28 In some organisms, the disaccharide trehalose is accumulated at high concentrations (0.5 M) when under stress.29–31 Trehalose (a non-reducing carbohydrate) has been amply demonstrated to be both a highly effective stabilizer against both freeze-drying and heat inactivation of enzymes.32–34 Trehalose stabilizes the structure of enzymes by hampering the structural dynamics leading to unfolding through increasing the viscosity of the medium and by compacting the protein native state.34–36 The efficiency of trehalose is based on its inert chemical structure and to the interaction with water molecules at the protein surface.37–40 Trehalose-protein interaction favors the compactness of the folded state of proteins while preventing structural



motions (through increasing the viscosity of the medium) away from the native state.36,37 However, trehalose presence also leads to the inhibition of some enzymes through the same mechanism described for protein stabilization.41,42 Therefore, it has been used to study enzyme catalysis in structural-dynamics dependent reactions.32,35,41 The relationship of enzyme structural dynamics in catalysis with the viscosity of the medium (generated by trehalose) has been studied by using Kramers´ theory.35,43,44 This theory allows the analysis of enzyme activity in viscous solutions by evidencing the presence or not of diffusive structural components in the catalytic mechanism of enzymes.32,41,42 In this work, LDH kinetics (pyruvate reduction to lactate) was studied in the absence and presence of the disaccharide trehalose and at different temperatures. The dependence of kinetics parameters upon temperature was determined first in the absence of trehalose: Vmax increased exponentially and Km displayed a sigmoid transition toward a low-affinity value similar to that of protein unfolding. Interestingly, physiological concentrations of trehalose did inhibit LDH catalysis as Vmax decreased in its presence. By contrast, trehalose presence increased LDH affinity (by decreasing Km) and also prevented the temperature-mediated transition toward the low-affinity state. Notably, the maximum catalytic rate (Vmax) was inversely dependent on the viscosity of the medium as described by Kramers´ theory.35,43 As result, LDH activation energy (Ea) for catalysis was increased. LDH catalytic cycle involves an important structural motion, probably that of the loop closing the active site as suggested by molecular dynamics simulation.7,10,45 Further, trehalose increased catalytic efficiency by increasing LDH affinity probably through the compaction and promotion of active site order.


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EXPERIMENTAL SECTION Materials. Lactate dehydrogenase (LDH) type II from rabbit muscle suspended in ammonium sulfate was acquired from Roche Diagnostic Corp. (Indianapolis, IN. USA). Sodium pyruvate, trehalose, NADH, 1,4-piperazinediethanesulfonic acid (PIPES) were from Sigma-Aldrich® (Merck KgaA, Darmstadt, Germany). Electrophoresis reagents and Bradford kit for protein concentration determination and Coomassie Blue R-250 were from BioRad® Laboratories, Inc. (Hercules, CA. USA). All other reagents were of the highest quality available commercially. Lactate dehydrogenase (LDH) purification. LDH suspension in ammonium sulfate salt (3.2 M) contains minor protein contaminants as evaluated by SDS-PAGE and visualized by Coomassie Blue R-250 staining. LDH was further purified as follows: ammonium sulfate salt (3.2 M) was removed from the LDH suspension by using PD10 desalting column® from GE Healthcare Bio-Sciences AB (Uppsala, Sweden), LDH and its protein contaminants were eluted with buffer PIPES 10 mM, pH 7.0. Then, protein contaminants were removed by ion exchange chromatography using a 5 mL HiTrap Q HP column from GE Healthcare Bio-Sciences AB, and LDH eluted with a linear concentration gradient of NaCl (1M stock solution in buffer PIPES 10 mM, pH 7.0). LDH elution was monitored by absorbance at a wavelength of 280 nm. Fractions containing LDH were pooled and then, desalted with a PD10 desalting column®. LDH concentration was determined by the Bradford assay using γ-globulin as standard.46 Protein purity (>95% by densitometry) was verified by SDS-PAGE and Coomassie Blue R-250 staining (Figure S1). LDH activity assay. LDH activity was assayed as described by Demchenko et al.,47 briefly: LDH (0.54 µg) was suspended in 2 mL of buffer PIPES 10 mM (pH 7.0), containing NADH 200 µM, pyruvic acid (0.1 - 1.2 mM), and trehalose (0 - 0.8 M) as indicated. NADH oxidation



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coupled to pyruvate reduction was followed spectrophotometrically at a wavelength (λ) of 340 nm. The rate of LDH reaction was calculated from the slope of the linear portion by using a molar extinction coefficient (ε) of 6,200 M-1cm-1. Dynamic viscosity measurement. A falling ball type viscometer (Gilmont Instruments Inc., Barrington, Il), mounted in a constant-temperature chamber was employed as described by Uribe and Sampedro.42 Trehalose solutions (0 – 0.8 M) were prepared in 10 mM PIPES, pH 7.0 and dynamic viscosity (cP) determined at different temperatures (25 – 35 °C). Before the measurement of ball descent, trehalose solutions were degassed and allowed to equilibrate at each temperature of test for 10 min. Data analysis. The velocity (v) of NADH oxidation (pyruvate reduction) was plotted against the concentration of pyruvate (S). Then, the data were fitted to the Michaelis-Menten equation (Eq. 1) by non-linear regression using the iterative program Microcal Origin 6.0® (Microcal Software Inc. MA). v = Vmax · S / Km + S

(1)

where Vmax is the maximum velocity, which is proportional to the rate constant of catalysis (kcat) relative to the total concentration ([Et]) of the enzyme (Vmax = kcat[Et]), Km is the MichaelisMenten constant, or the substrate concentration when v = 0.5Vmax. LDH efficiency at a given condition was calculated as the quotient kcat/Km. The activation energy (Ea) for pyruvate reduction by LDH was calculated from the slope value of the generated straight line by linear regression using the logarithmic form of the Arrhenius equation (Eq. 2) plotting ln Vmax versus 1/T. ln Vmax = ln A - (Ea/RT)

(2)

where R is the gas constant (8.315 J/molK), T is the absolute temperature and A is a constant.


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The dynamic viscosity of trehalose solutions at the indicated temperature (25 - 35°C) was determined by using the time of ball descent and equation 3 (Eq. 3) as described elsewhere.41,42 η = K·(δb-δl)·t

(3)

where η is the solution viscosity in cP, K is the viscometer constant, δb is the density of the ball, δl is the density of the liquid and t is the time of ball descent. Falling ball viscometer was calibrated with pure water at different temperatures. The calculated water viscosities were similar to the universal accepted values48 (Figure S2). Standard deviation for trehalose viscosity data was less than 5%.41,42 Analysis of LDH reaction rates in trehalose solutions was performed according to Kramers´ theory.41,42,44,49 Kramers´ theory describes the rate of protein unfolding and enzyme catalysis (involving structural motions) relative to viscosity (friction) of the suspending medium.35,42 In enzyme catalysis, when reaction is coupled to the viscosity of the medium the rate constant (Vmax or kcat) shows an inversely direct linear relationship with viscosity as described in equation 4 (Eq. 4).35,43 Vmax = η-1 exp (∆U/RT)

(4)

where Vmax is the maximum rate of catalysis for a given enzyme, ∆U is the height of the potential energy barrier imposed by friction of the protein mobile structure with molecules of the medium,35,43 η is the viscosity of the medium, R is the gas constant (8.315 J (K·mol)-1) and T the absolute temperature. Enzyme catalysis dependence on viscosity is generally observed as a linear decrease in Vmax as viscosity (η) increases.35,50

RESULTS AND DISCUSSION Effect of temperature on LDH kinetics. LDH activity was assayed at the range of temperature from 25 to 35 °C as this seems to be the optimum for catalysis (Figure 1a).51–53 At 7 Environment ACS Paragon Plus


25 °C, LDH kinetics was hyperbolic with pyruvate as substrate (Figure 1a). Velocity data were analyzed by non-linear regression using the Michaelis-Menten equation 1 (Eq. 1, Figure 1a). The calculated kinetics parameters were Vmax = 355.5 ± 7.1 µmoles pyruvate(minmg prot)-1 and Km = 96.3 ± 0.8 µM pyruvate, these values were similar to those previously reported.54 Then, as assay temperature was increased (2.5 °C, stepwise), Vmax increased exponentially reaching a value at 35 °C of 618.5 ± 8.6 µmoles pyruvate(minmg prot)-1 (Figure 1a). Thus, the maximum rate of LDH catalysis doubled (Figure 1a) in agreement with the general observation that catalytic rate for enzymes doubles every 10 °C.55 However, at temperatures above 35 °C the increase of LDH activity began to deviate from the exponential pattern, the above probably because of a decrease in affinity (discussed below) and/or thermal inactivation (Figure 2a, inset). 53,54,56

The calculated Vmax was used to determine the activation energy (Ea) for pyruvate

reduction by using the Arrhenius equation (Eq. 2, Figure 2a), Ea = 43.09 ± 0.89 kJ/mol (Figure 2). The Ea value was in good agreement to that published (Figure 2a).57

(a)

(b)


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Figure 1. Effect of temperature on LDH kinetics. LDH activity (pyruvate reduction) was determined spectrophotometrically monitoring the absorbance of NADH at λ= 340 nm in a constant temperature cuvette chamber. The velocity of pyruvate reduction was calculated from the straight portion of the absorbance decay line formed. (a) LDH velocity data at temperatures: 25.0, 27.5, 30.0, 32.5 and 35.0 °C. Data velocities were fitted to the MichaelisMenten equation (Eq. 1) by non-linear regression. (b) Lineweaver-Burk plot of data in (a). Linear regression of the data is shown; r values were above 0.98 in all dataset. The x- and y-axis intercept values are -1/Km and 1/Vmax, respectively. The experiment was performed three times and standard deviation (SD) was less than 5%. LDH affinity for pyruvate (Km) decreased when assay temperature was increased (Figure 1b and Figure 2b). In the Lineweaver-Burk plot, the x-axis intercept (-1/Km) of the straight lines generated by linear regression of the data showed a displacement toward the origin (O) as temperature increased (Figure 1b). Km at 30 °C (149 µM pyruvate, Figure 2b) was in good agreement to that reported previously for LDH from rabbit muscle54,58 and above 35 °C, no further change was observed (data not shown).54 Notably, when plotted Km versus temperature a sigmoid pattern was observed (Figure 2b) evidencing a two-state binding site structure. Thus, suggesting thermal-modulation of LDH affinity for pyruvate (Figure 2b). The calculated transition temperature was 28.8 ± 0.6 °C. In agreement with this result, it has been reported that high temperature induces high flexibility in the whole LDH structure and a distortion of the catalytic pocket and its structural surroundings.51 Therefore, it was concluded that temperature modulates the structure of LDH pyruvate binding site, leading it to fluctuate between high- and low-affinity state at low and high temperature, respectively. Whether the temperature-mediated modulation of LDH affinity for pyruvate has a role in cellular metabolism, its energy status



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and/or temperature adaptation is a matter of future experiments/discussion.57,58 Nonetheless, it is already known that LDH from cod (Gadus morhua) displays low Km values (high affinity) for pyruvate at low temperatures.57

(a)

(b)

Figure 2. Effect of temperature on LDH kinetic parameters. (a) Arrhenius plot for LDH catalysis. The activation energy (Ea) was calculated from the slope value as described by the Arrhenius equation 2 (Eq. 2), Ea = 43.1 ± 0.9 kJ/mol. Inset, LDH Vmax at different temperatures. The data were fitted to a single exponential equation up to 35 °C. Vmax at temperatures above 35 °C were not included in the fitting in figure (a) because of thermal inactivation of LDH. (b) Plot of LDH Km versus temperature. Km data were fitted by non-linear regression to the Boltzmann equation. The calculated temperature for Km transition was 28.8 ± 0.6 °C.

: ↔ : ↔ : : !"

(5)

In LDH, the formation of the catalytic complex LDH:NADH:pyruvate has been of great interest thus amply studied.9,10,59–61 A kinetic mechanism has been proposed (Eq. 5).60 The


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present study on LDH kinetics dependence to temperature provides some insight about such process, as it is known that the active site in enzymes is the most thermally-sensitive structural element,62 e.g., it has been reported in psychrophilic enzymes that activity is usually lost before the protein becomes denatured.62 Local structural changes take place in LDH before (K1 in Eq. 5) and after binding of pyruvate (K2 in Eq. 5).10,59 In this regard, studies on oxamate (a pyruvate analogue) binding to binary complex LDH:NADH using temperature jump infrared spectroscopy showed that temperature increase (from 20 to 35 °C) does not favor the formation of the LDH:NADH:pyruvate complex.59 More recently, studies on LDH (from rabbit muscle) using neutron spin echo spectroscopy in conjunction with molecular dynamics simulations showed that increasing temperature leads to distorting the catalytic site and its structural surroundings.51 Thus demonstrating that thermally induced structural changes in LDH active site dissipates to nonproductive states (probably LDH:NADHclosed in Eq. 5)51 compromising catalysis. The present result suggested that LDH:NADHclosed complex harbors a partially unordered/unfolded binding site displaying low affinity for pyruvate. Certainly, this suggestion does not mean a large-scale unfolding of pyruvate binding site but rather subtle variations in local structure and water arrangement.10 Effect of trehalose on the rate of catalysis of LDH. The rate-limiting step for LDH turnover (kcat) has been proposed to be the movement of closure of a loop over the binding pocket after complexation of pyruvate.10,63 Currently, it is being recognized that motions of active site loops in enzymes may have an essential role in catalysis.64 Therefore, it was hypothesized that the disaccharide trehalose may modulate LDH catalytic step (kcat).



(a)

(b)

Figure 3. Effect of physiological concentrations of trehalose on LDH kinetics. The LDH assay reaction included trehalose at concentrations of 0, 0.2, 0.4, 0.6 and 0.8 M. (a) and (b) LDH kinetics in the presence of trehalose at 25 and 35 °C, respectively. LDH velocity data were fitted by non-linear regression to Michaelis-Menten equation 1 (Eq. 1). LDH kinetics was studied in the presence of physiological concentrations of trehalose30 and at different temperatures. At 25 °C, the LDH catalytic rate (velocity) decreased by the presence of trehalose (Figure 3a). Velocity data were fitted by non-linear regression to the Michaelis-Menten equation 1 (Eq. 1) and kinetic parameters calculated. LDH kinetics in the presence of trehalose was hyperbolic, except that Vmax decreased as trehalose concentration increased (Figure 3a). Trehalose effect on LDH catalysis (Vmax) decreased as assay temperature increased thus at 35 °C, Vmax was only slightly modified (Figure 3b). Inhibition of enzyme catalysis (Vmax) by trehalose has been reported before on the plasma membrane H+-ATPase from the yeast Kluyveromyces lactis.41 Similarly, it has been reported that dextran (a complexly branched glucan) inhibits the activity of LDH and yeast alcohol dehydrogenase (YADH).65,66


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Kramers´ theory and trehalose-mediated LDH inhibition. Trehalose effect on LDH catalysis (Vmax) decreased as assay temperature increased thus at 35 °C Vmax was only slightly modified (Figure 3b). The Vmax temperature behavior in the presence of trehalose suggested the probable mechanism of inhibition, namely by friction with molecules of the medium (trehalose and water) or viscosity-mediated. As mentioned above, it has been proposed that the motion of a structural loop near the binding site is essential for catalysis.10 Thus, it seems plausible that the observed inhibition of LDH activity in the presence of trehalose may be due to hampering the movement of this structural loop by viscosity.35,41As temperature rises, thermal energy becomes available thus allowing the motion of the loop over the LDH active site.67 Kramers´ theory has been used to analyze protein folding-unfolding49,68 and enzyme kinetics in viscous solutions.35,41,42,44 It has been demonstrated that viscosity (η) modulates the rate of enzyme reactions when a structural diffusive catalytic step is present.35,43 In order to test the above hypothesis, first it was determined the viscosity of trehalose solutions at all concentrations and assay temperatures.42 Then, Vmax was plotted versus the inverse of solution viscosity (η-1) as stated in equation 4 (Eq. 4, Figure 4a). The plot shows clearly that the maximum catalytic rate (Vmax) of LDH was dependent on viscosity (η) of the medium at all temperatures tested (Figure 4a). This result provides experimental support to the proposal that a diffusive structural motif in LDH is essential for catalysis (Figure 4b).10,63 The involvement of others mechanisms such as a direct and specific interaction of trehalose molecules with the binding pocket (water-trehalose hydrogen bond replacement)69 or the indirect effect of the trehalose coating on the structure and dynamics of water in the ligand pocket domain (trapped water layer)70,71 may be ruled out, as molecular dynamics simulation neither shows that trehalose (at 0.5M) removes water from protein surface nor establishes hydrogen bonds with the protein.72 Therefore, it was concluded



that trehalose modulates the rate of LDH catalysis through the viscosity of the medium (Figure 4a).

(a)

(b)

Figure 4. (a) LDH Vmax dependence on the viscosity of the medium. The viscosity (η) of trehalose solutions (0, 0.2, 0.4, 0.6 and 0.8 M) was determined by using a falling ball viscometer as described in methods. LDH Vmax in the presence of trehalose was determined as described in Figure 3 at the following temperatures: 25.0, 27.5, 30.0, 32.5 and 35.0 °C. Linear regression of the data was performed by using the modified Kramers´ equation 4 (Eq. 4). (b) Proposed hampering of the motion of the LDH catalytic loop by increased the viscosity of the medium (η) generated by trehalose. The amino acid residues involved in pyruvate binding (H192, N137, R168, and T247) and catalysis (R105) are in sticks in the three-dimensional (3D) structure of the LDH (PDB entry 3H3F) monomer. After pyruvate binding, the catalytic loop (amino acid residues 98 – 110) moves over the active site to promote hydrogen transfer via polarization of carbonyl group of pyruvate by R105.


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Figure 5. Effect of trehalose on the activation energy (Ea) for pyruvate reduction by LDH. Arrhenius plot was generated by using the calculated Vmax at different trehalose concentrations: 0, 0.2, 0.4, 0.6 and 0.8 M. The activation energy (Ea) was calculated from the line slope value (m = -Ea/R) by linear regression of the data according to Eq. 2. Inset, dependence of Ea on trehalose concentration. Ea data were fitted by nonlinear regression to a single exponential equation. Effect of trehalose on LDH activation energy (Ea). The Ea for LDH catalysis was determined by using the Arrhenius equation (Eq. 2) (Figure 5). Trehalose led to increasing exponentially the energy barrier for pyruvate reduction (Figure 5, inset) from Ea = 43.1 to 66.0 kJ/mol in the absence and presence of 0.8 M trehalose, respectively. The increased energy barrier for catalysis probably resulted from the trehalose-mediated hampering of catalytic loop movement over the active site (Figure 4b).37 Therefore, transition state formation and stabilization become less probable in LDH when viscosity increases (Figure 4 and Figure 5, inset). Trehalose is a matrix forming carbohydrate when water content is low.71 Therefore, under



this condition LDH activity would cease because of the high energy barrier imposed for pyruvate reduction (Figure 5 and inset).

Figure 6. Effect of trehalose on LDH Km at different temperatures. LDH kinetics was performed as described in Figure 3 in the presence of different trehalose concentrations and temperatures. Km values were calculated by fitting the data by nonlinear regression to Michaelis-Menten equation (Eq. 1). Effect of trehalose on pyruvate affinity in LDH. Km decreased in the presence of trehalose thus LDH displayed a higher affinity for pyruvate than in its absence (Figure 6). The effect on Km seems to be due to the increase in the compactness of the binding site.36 Trehalose probably promoted the best active site structural arrangement similar to that observed in glucose oxidase from Aspergillus niger.32 It is known that the effect of trehalose on protein structure is associated with the slowing down of water dynamics around the disaccharide moiety,73 which results from the stable trehalose-water interaction. Trehalose also slows down the dynamics of protein hydration waters,74 the above due to the coupling of protein surface, water, and trehalose.71,75


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Nonetheless, the present results on Km suggested that trehalose favored the equilibrium toward a compact ordered state of the binding site (LDH:NADHopen) thus, facilitating the binding of pyruvate (K2 in Eq. 5) (Figure 6).76 In this regard, LDH active site appears highly hydrophilic when analyzed with Kite and Doolittle77 hydropathy criteria (results not shown). Further, two disordered segments were identified when LDH amino acid sequence was analyzed as described by Linding et al. (Figure 7a).78 Notably, amino acid residues between 189 and 201 form an intrinsically disordered loop (Figure 7a), where the histidine residue (H192) involved in pyruvate binding and complex stabilization is located (Figure 7b). Therefore, it seems probable that trehalose decreased the fluctuations of the disordered loop favoring the proper arrangement of the binding site (Figure 7b) and as consequence enhancing pyruvate affinity (Figure 6), i.e., structural dynamics simulation showed that the transition from the LDH:NADHclosed to LDH:NADHopen states (K1 in Eq. 5) involves changes in solvation and hydrogen bonds rearrangements in pyruvate binding site.10 In agreement to the above suggestion, Tsai et al.67 stated that the number of conformations a protein may access reduces significantly in a viscous solvent.67 In addition, it has been observed that trehalose increases the binding affinity for glucose in human glucokinase by shifting the protein conformation to a condensed state.79 Furthermore, Das et al.36 showed that trehalose decreases the hydrodynamic radius of BSA (compacting its structure) leading to decrease its ligand binding capacity.36 Effect of trehalose on catalytic efficiency of LDH. In the absence of trehalose, LDH affinity for pyruvate decreased when temperature was increased (Figure 1b and Figure 2b), thus diminishing the enzyme catalytic efficiency (Figure 8).51 Interestingly, LDH catalytic efficiency seems to be higher at lower temperatures, e.g. at 25 °C (Figure 8). By contrast, in the presence of trehalose (0.2 - 0.8 M), Km was lower than in its absence and kept nearly constant regardless of



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assay temperature (Figure 6). In spite that trehalose decreased LDH Vmax (Figure 3), catalytic efficiency was higher in its presence (Figure 8) because of the effect on Km (Figure 6). Therefore, it was concluded that trehalose increased LDH catalytic efficiency (Figure 8) by favoring the formation of the competent (LDH:NADHopen) complex for pyruvate binding. Similarly, catalytic efficiency of glucose oxidase from Aspergillus niger is enhanced in the presence of trehalose.32

(a)

(b)

Figure 7. (a). Plot of order/disorder of the LDH structure. LDH amino acid sequence was analyzed for globularity and disorder by using the online software GlobPlot

2.3

(http://globplot.embl.de/ plot.embl.de/).78 Disordered segments (grey area) corresponds to amino acids sequences 154 – 161 and 189 – 201. (b). Pyruvate binding site in LDH (PDB entry 3H3F) monomer. Amino acid residues involved in pyruvate binding (N137, R168, H192, and T247) and catalysis (R105) are shown in sticks. Disordered (loop) amino acid segment (189 – 201) harboring pyruvate-interacting residue H192 is colored in red.


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Figure 8. Effect of trehalose and temperature on LDH catalytic efficiency. LDH kinetics parameters were calculated as described in Figure 3. CONCLUSIONS The present results provide experimental evidence supporting the proposal that catalysis of LDH depends on the motion of a structural loop above the active site10,63 and that trehalose may hinder such motion via increased solution viscosity thus resulting in modulation of LDH activity as described by Kramers´ theory (Figure 4a).41,47 The mechanism of protein stabilization by trehalose has been claimed to be due to its effects on protein compactness and structural dynamics.34,69,80,81 Structural stability and catalysis/function in LDH seems to be intimately related to global and local flexibility,82 external and internal fluctuations, and other dynamic processes of the suspending medium like viscosity. The present results provide experimental evidence that trehalose may interplay also in LDH function through the same mechanism of



protein stabilization (Figure 6 and 8), namely by promoting the structural order of pyruvate binding site and hindering the transition from LDH:NADHopen to LDH:NADHclosed state. The above may be relevant in some organisms during diauxic curve transition, entrance to quiescence, high and low temperature, and/or desiccation.29,83,84 Therefore, enzyme catalysis optimization/adaptation to a given temperature probably results from modulation of flexibility of binding site62 whether via amino acid mutations or modification of physicochemical properties of the aqueous media. Finally, Kramers´ theory provides the scientific interpretation and the mathematical expression to analyze the effect of compatible solutes in enzyme function or the behavior of enzymes in viscous solutions.44

AUTHOR INFORMATION Corresponding Author *E-mail: [email protected]. Phone number: +52(444) 8262-300, ext.: 5715 Author Contributions The manuscript was written by J.G.S. J.M.H.-M. performed the experiments. J.G.S analyzed the experiments. All authors have given approval to the final version of the manuscript. Notes The authors declare no competing financial interest.


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ACKNOWLEDGMENTS Partially supported by SEP-México (Fortalecimiento de Cuerpos Académicos 2017 – Interacciones Moleculares en Sistemas Biológicos, grant number UASLP-CA-263) and CONACYT-México (Fronteras de la Ciencia Reformulación de la Termodinámica y Estadística y la Materia fuera de Equilibrio, grant number FC-1155-2016). J.M.H-M was a recipient of a CONACYT fellowship.

ASSOCIATED CONTENT Supporting Information Available Figure S1. SDS-PAGE of purified lactate dehydrogenase (LDH) from rabbit muscle (PDF). Figure S2. Plot of water viscosity versus temperature (falling ball viscometer calibration) (PDF). This information is available free of charge via the Internet at http://pubs.acs.org.

ABBREVIATIONS LDH, lactate dehydrogenase; YADH, yeast alcohol dehydrogenase; NADH, Nicotinamide adenine dinucleotide reduced form; Ea, activation energy; SD, standard deviation; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; PIPES, 1,4piperazinediethanesulfonic acid.



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TOC Graphic


Trehalose Mediated Inhibition of Lactate Dehydrogenase from Rabbit

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