Trehalose-Mediated Inhibition of the Plasma Membrane H+-ATPase from Kluyveromyces lactis: Dependence on Viscosity and Temperature

The effect of increasing trehalose concentrations on the kineticsof the plasma membrane H⁺-ATPase from Kluyveromyces lactis wasstudied at different temperatures. At 20°C, increasing concentrationsof trehalose (0.2 to 0.8 M) decreased V_max and increased S_0.5(substrate concentration when initial velocity equals 0.5 V_max),mainly at high trehalose concentrations (0.6 to 0.8 M). Thequotient V_max/S_0.5 decreased from 5.76 µmol of ATP mgof protein^-1 min^-1 mM^-1 in the absence of trehalose to 1.63µmol of ATP mg of protein^-1 min^-1 mM^-1 in the presenceof 0.8 M trehalose. The decrease in V_max was linearly dependenton solution viscosity ( ${eta}$ ), suggesting that inhibition was dueto hindering of protein domain diffusional motion during catalysisand in accordance with Kramer's theory for reactions in solution.In this regard, two other viscosity-increasing agents, sucroseand glycerol, behaved similarly, exhibiting the same viscosity-enzymeinhibition correlation predicted. In the absence of trehalose,increasing the temperature up to 40°C resulted in an exponentialincrease in V_max and a decrease in enzyme cooperativity (n),while S_0.5 was not modified. As temperature increased, the effectof trehalose on V_max decreased to become negligible at 40°C,in good correlation with the temperature-mediated decrease inviscosity. The trehalose-mediated increase in S_0.5 was similarat all temperatures tested, and thus, trehalose effects on V_max/S_0.5were always observed. Trehalose increased the activation energyfor ATP hydrolysis. Trehalose-mediated inhibition of enzymesmay explain why yeast rapidly hydrolyzes trehalose when exitingheat shock.

INTRODUCTION

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Introduction

When yeast cells are subjected to stress conditions such asdehydration, high salt concentrations, or high temperatures,the trehalose-synthesizing pathway is activated and cytoplasmictrehalose rises to 0.5 M and above (16-18, 38). It has beendemonstrated previously that under these stress conditions trehalosestabilizes the structure of membranes and proteins as well asthe activity of enzymes (8, 16, 34, 35). As soon as the stresscondition is over, trehalose is rapidly hydrolyzed (18), suggestingthat the accumulation of this disaccharide may not be compatiblewith certain cell functions (43).

The properties of trehalose in solution might explain both themechanism of protection observed when the cell is under stressand the need to rapidly hydrolyze the disaccharide once conditionsreturn to normal (26, 32). It has been reported previously thattrehalose promotes the preferential hydration of protein surfaces(13) and favors the most compact state of the protein, inhibitingintramolecular motions that might result in denaturation, i.e.,trehalose confers thermal stability to proteins (4, 34) as evidencedby the increase in their transition temperature (24). However,proteins are dynamic molecules that often need to undergo conformationalchanges while performing specific functions such as enzymaticcatalysis or ligand binding (12, 30), and therefore, it hasbeen proposed elsewhere that those enzymes undergoing majorconformational changes during their catalytic cycle are inhibitedby viscogenic agents such as polyols (9), as protein backbonemotions would be hindered in a highly viscous solution (30).In studies of carbon-monoxy-myoglobin, a trehalose-mediatedinhibition of carbon monoxide release upon flash photolysishas been reported (15). The mechanism underlying inhibitionhas been proposed to be the trehalose-mediated hindering ofprotein conformational changes (6, 15). Additionally, it hasbeen demonstrated elsewhere that trehalose reduces the motionalfreedom of the myoglobin loop and helix structures by forminga tight hydrogen bond network around the protein (7). A secondeffect of trehalose, also related to immobilization of proteinstructures, seems to be the inhibition of the heat shock protein-catalyzedrefolding of partially denatured proteins, observed immediatelyafter a period of stress (39). It has been proposed that a highconcentration of trehalose would stabilize the partially denaturedconformation of proteins, interfering with the refolding catalyzedby the heat shock proteins (39).

E₁E₂-ATPases undergo major conformational changes during theircatalytic cycle, alternating between a nonphosphorylated E₁state and a phosphorylated E₂ state (10). The structures ofstates E₁ and E₂ are different, as suggested by their sensitivityto proteases and antibodies (10, 29). The Ca²⁺-ATPase in thesarcoplasmic reticulum, whose three-dimensional structure hasbeen defined elsewhere (41); the plasma membrane Na⁺-K⁺-ATPase,which is an antiporter (19); and the H⁺-ATPases from the plasmamembrane of plants and fungi (EC 3.6.1.35) (37) are E₁E₂-ATPases(10). The H⁺-ATPase from the plasma membrane of diverse fungiand yeasts is the primary pump needed to generate the protongradient that energizes all secondary active transport in andout of the cell (33, 37). The physiological significance ofH⁺ transport in yeast is illustrated by the lethality of thedeletion of the gene encoding the H⁺-ATPase (36). Furthermore,during heat shock a high H⁺-ATPase activity is required to maintainthe ionic gradients and active transport activities across theplasma membrane, which undergoes an increase in permeabilityas temperature rises (5).

The inactivation effects of different stress conditions on theKluyveromyces lactis H⁺-ATPase have been tested, and the inactivationkinetics have been reported elsewhere (34, 35). During freeze-drying,the H⁺-ATPase is denatured unless high concentrations of monosaccharidesor disaccharides are present, trehalose being the most effectiveprotective agent (35). In similar studies of the thermoinactivationof the H⁺-ATPase, it was observed that the temperature rangein which full protection by trehalose occurs is that at whichcytoplasmic trehalose accumulation enhances yeast survival duringheat shock (34).

It was decided to analyze whether trehalose inhibited the isolatedK. lactis H⁺-ATPase at different temperatures. We found that,at 20°C, trehalose inhibited the activity of the H⁺-ATPasemainly through a decrease in V_max, which was linearly dependenton solvent viscosity as predicted by Kramer's theory (22). Inaddition, sucrose and glycerol exhibited the same viscosity-inhibitoryactivity correlation as trehalose. As temperature increased,the ATPase activity increased exponentially while the trehaloseeffect on V_max diminished in proportion with the decrease intrehalose solution viscosity. Enzyme inhibition at low temperaturesmay explain why, at 30°C and below, yeast cells do not accumulatetrehalose and why these cells rapidly hydrolyze the accumulatedtrehalose as soon as they exit a heat shock period. In contrast,at heat shock temperatures ATP hydrolysis is not inhibited.

MATERIALS AND METHODS

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Materials and Methods

Materials. Trehalose, ATP-disodium salt, pyruvate kinase type II from rabbitmuscle, lactate dehydrogenase type XI from rabbit muscle, sodiumazide, NADH, N-tetradecyl-N,N-dimethyl-3-ammonium-1-propanesulfonate(Zwittergent 3,15), and phosphoenolpyruvate mono-(cyclohexylammonium)salt were from Sigma Chemical Co. (St. Louis, Mo.). Zymolyase-20Twas from ICN Pharmaceuticals Inc. (Costa Mesa, Calif.). Allother reagents were of the best quality available commercially.

Enzyme purification. Isolation was performed essentially as described before (14).Briefly, the yeast K. lactis strain WM27 was grown in yeastextract-peptone-dextrose medium at 30°C for 20 h. Then,cells were harvested at mid-log phase by centrifugation. Yeastcells were incubated at 30°C in 1 M sorbitol, pH 7.0, containingzymolyase-20T (20 U/g [wet weight]) for 1 to 2 h until spheroplastswere detected by the decrease in turbidity of an aliquot (50µl) immersed in distilled water (3 ml). Spheroplasts weredisrupted by sonication, and the plasma membrane was obtainedby differential centrifugation (14). The H⁺-ATPase was purifiedfrom the plasma membrane as described by Bowman and Slayman(3) and modified by Guerra et al. (14). Briefly, the plasmamembrane was diluted to 2 mg/ml in buffer A (0.6 M KCl, 75 mMTris, 6 mM EDTA, pH 7.2)-1 mM EGTA-0.09% (wt/vol) deoxycholate.The mixture was incubated for 10 min at 4°C and then centrifugedat 4°C and 100,000 x g for 1 h. The pellet was resuspendedin 10 ml of buffer B (0.3 M KCl, 25 mM Tris [pH 7.5], 45% [vol/vol]glycerol, and 2 mM EDTA), and then the protein concentrationwas measured and adjusted to 5 mg/ml in the same buffer B. Azolectinwas added to a final concentration of 5 mg/ml, and Zwittergent3,14 was slowly added to a final Zwittergent/protein ratio of0.85 (wt/wt). The suspension was homogenized and centrifugedat 4°C and 100,000 x g for 1 h. The supernatant was subjectedto centrifugation at 100,000 x g for 14 h on a discontinuousglycerol gradient (65, 60, 55, and 50% [vol/vol] glycerol in10 mM Tris [pH 7.0], 1 mM EDTA, 0.1% cholate, 1 mg of azolectin/ml).A Beckman ultracentrifuge and 60 Ti fixed-angle rotor were used.The lower half of the supernatant and the pellet were recovered,diluted twofold, and centrifuged at 100,000 x g for 2 h. TheATPase was suspended from the pellet in a small volume of 1mM EGTA-Tris, pH 7.0, and kept at -70°C until used. On sodiumdodecyl sulfate-polyacrylamide gel electrophoresis, the 100,000-M_rband corresponding to the plasma membrane H⁺-ATPase was about50% of the total protein yield. The H⁺-ATPase specific activityin this preparation, measured at 30°C, was 18.9 ±0.3 µmol of ATP mg of protein^-1 min^-1. Protein concentrationwas determined by the method of Lowry et al. (25) with bovineserum albumin as standard.

Measurements of H⁺-ATPase activity. The ATP saturation kinetics of the plasma membrane H⁺-ATPasewere evaluated as described elsewhere for the Ca²⁺-ATPase fromsarcoplasmic reticulum (41), by using the enzyme-coupled assaydescribed by Anderson and Murphy (2). The assay medium (2 ml)was 10 mM PIPES [piperazine-N,N'-bis(2-ethanesulfonic acid);pH 7.0], 80 mM KCl, 5 mM sodium azide, 5 mM phosphoenolpyruvate,200 µM NADH, 12.5 IU of pyruvate kinase, 10.45 IU of lacticdehydrogenase, and 5 mM MgCl₂. Increasing concentrations ofATP (0.25 to 5.0 mM) and trehalose (0 to 0.8 M) were added asindicated. The assay solution was incubated at the indicatedtemperature for 10 min, and the ATPase was added (4.3 µgof protein in 4 µl) to start the reaction. The absorbancedecay at 340 nm was recorded continuously in an Aminco DW2000double-wavelength spectrophotometer in split mode, equippedwith a cell with a thermostat. Initial velocities of ATP hydrolyzed(expressed as micromoles of ATP milligram of protein^-1 minute^-1)were calculated from the slope of the linear portion of eachtrace, with an NADH extinction coefficient of 6,200 M cm^-1.The experiment was performed at temperatures between 20 and40°C as indicated. Additionally, it was determined thatthe coupled enzyme assay was not affected by temperature orby trehalose and, thus, the observed variations in rate resultedspecifically from the enzyme activity response to the concentrationof ATP.

Viscosity measurements. A falling-ball-type viscometer (Gilmont Instruments, Barrington,Ill.) mounted in a constant-temperature chamber was employed.Trehalose solutions (0 to 0.8 M) were prepared in 10 mM PIPES,pH 7.0. The viscometer was filled with each trehalose solution,degassed by vacuum application, and allowed to equilibrate ateach temperature for 10 min. Once the assay temperature wasreached, the time of ball descent was measured and used to calculatethe viscosity (26) by using equation 1:

(1)
where ${eta}$ is the viscosity in centipoise, K is the viscometer constant, ${delta}$ _b is the density of the ball, ${delta}$ _l is the density of the liquid,and t is the time of ball descent. Experimental data were reproducible,and standard deviations were less than 5%.

Data analysis. Initial velocities of ATP hydrolysis were plotted against theconcentration of ATP. The iterative program Microcal Origin6.0 (Microcal Software Inc., Northampton, Mass.) was used toanalyze the data by nonlinear regression. The Hill equation(equation 2) was used in the fitting of the initial velocitydata:

(2)
where v is the initial velocity,V_max is the maximum velocity, S is the concentration of thevaried substrate, S_0.5 is the substrate concentration when v= 0.5 V_max, and n is the Hill number. From the kinetic parametersobtained with equation 2, the quotient V_max/S_0.5 was calculated.This is an estimate of the efficiency of the enzyme.

The energy of activation (E_a) for the hydrolysis of ATP wascalculated by using the logarithmic form of the Arrhenius equation(equation 3) by plotting log V_max versus 1/T and calculatingthe E_a from the slope value:

(3)
whereR is the gas constant (8.314 J K^-1 mol^-1), T is the absolutetemperature, and A is a constant.

The dependence of solution viscosity on solute concentrationexpressed as mass fraction c (0 < c < 1) (23) was analyzedby using the treatment described by Mooney (28). Thus, the equationwas as follows:

(4)
where ${eta}$ is the macroscopicviscosity of the solvent, A is characteristic of the solutehydrodynamic interactions, and B is proportional to a self-crowdingfactor.

Kramer's theory (22) was used to analyze the possible mechanismof inhibition. This theory describes the effect of solvent propertieson the behavior of protein reactions involving domain diffusionalmotions. In a reaction evolving from the substrate S, throughthe activated state X, to the product P (S $->$ X $->$ P), the rate at whichthe activated state is reached and the product is obtained isinhibited by the friction of the protein with the solvent. Inturn, friction is a function of viscosity ${eta}$ . Thus, the reactionrate constant depends linearly on ${eta}$ (equation 5) as describedby Jacob and Schmid (21):

(5)
wherek is the rate constant for the reaction (V_max for enzyme-catalyzedreactions), ${Delta}$ U is the height of the potential energy barrier,R is the gas constant, and T is the absolute temperature. Toassess whether the reaction performed by the H⁺-ATPase followsKramer's relation, the relative V_max data, defined as V_max0/V_max,were plotted against the relative viscosity, defined as ${eta}$ / ${eta}$ ₀,where V_max0 and ${eta}$ ₀ are the V_max and the viscosity in the absenceof trehalose, respectively, and V_max and ${eta}$ are the observed valuesat each trehalose concentration assayed (20).

RESULTS

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Results

ATP saturation kinetics of the K. lactis plasma membrane H⁺-ATPase: effect of trehalose. Initial velocity rates were obtained at 20°C and in thepresence of an ATP concentration ranging from 0.4 to 4 mM. Experimentswere performed in the absence and in the presence of differenttrehalose concentrations. Data were fitted to the Hill equationby nonlinear regression (Fig. 1A ). Under our experimental conditions,the rate of ATP hydrolysis displayed a sigmoid dependence uponATP concentration (Fig. 1A). The estimated kinetic parametersfor the enzyme are listed in Table 1. In the absence of trehalose,these parameters were similar to those reported elsewhere forthe H⁺-ATPases from Neurospora crassa and Saccharomyces cerevisiae(29, 33), as expected from the high degree of identity observedin the gene (PMA1) coding for the plasma membrane H⁺-ATPasein these organisms (27, 31). Trehalose inhibited the H⁺-ATPase,mainly through a decrease in V_max (Table 1). S_0.5 values increasedslowly at the lower trehalose concentrations tested (0.2 to0.4 M). However, at the higher trehalose concentrations tested(above 0.5 M) S_0.5 values varied more steeply (Table 1). Thedecrease in V_max plus the increase in S_0.5 resulted in a decreasein V_max/S_0.5 compared to that of the control: 2.4- and 3.6-foldlower at 0.6 and 0.8 M trehalose, respectively (Table 1). Incontrast, the estimated Hill number values (n) were not modified,indicating that the mechanism of cooperativity was not sensitiveto trehalose (Table 1).

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FIG. 1. Trehalose-mediated inhibition of the plasma membrane H⁺-ATPase. Initial rates of ATP hydrolysis were measured at 20°C as described in Materials and Methods. The reaction mixture contained 10 mM PIPES (pH 7.0), 80 mM KCl, 5 mM sodium azide, 5 mM phosphoenolpyruvate, 200 µM NADH, 12.5 IU of pyruvate kinase, 10.45 IU of lactic dehydrogenase, 5 mM MgCl₂, and the indicatedconcentrations of ATP; the final volume was 2 ml. The H⁺-ATPase (4.3 µg of protein in 4 µl) was added to start the reaction. (A) Plot of ATP hydrolysis versus ATP concentration at the following molar concentrations of trehalose: 0 ( ${circ}$ ), 0.2 (•), 0.4 ( ${square}$ ), 0.5 ( ${blacksquare}$ ), 0.6 ( ${triangleup}$ ), and 0.8 ( ${blacktriangleup}$ ). The points are the means of three independent experiments. In all cases, the standard deviation was less than 5%. The lines are the result of the best fit to the Hill equation (equation 2) by nonlinear regression. (B) Lineweaver-Burk plot of the data in panel A; the lines were obtained by linear regression of the data. (C) Replots of the slope ( ${circ}$ ) and the 1/v axis intercept (•) in panel B. The lines are the best fit to the equation y = a + b·exp(c·x), where a is the ordinate, b and c are constants, and x is the trehalose concentration.

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TABLE 1. Kinetic parameters of the H⁺ -ATPase activity assayed at different temperatures and in the presence of different concentrations of trehalose^a

The data in Fig. 1A are depicted in Fig. 1B as a plot of 1/vagainst 1/[ATP]ⁿ (Fig. 1B), where n is the Hill number valueestimated as described above, by nonlinear regression of thedata obtained at 20°C and at each trehalose concentration.The lines intersect at the left of the 1/v axis, indicatingthe mixed nature of the trehalose-mediated inhibition of theenzyme. In Fig. 1C, the slope (_appS_0.5ⁿ/_appV_max) and 1/v intercept(1/_appV_max) values were replotted against the concentrationsof trehalose. Remarkably, the lines observed in Fig. 1C werenonlinear and, instead, bent upward as trehalose concentrationincreased; this might have been expected, since trehalose doesnot seem to bind to a specific site in the enzyme and, therefore,its effects cannot be analyzed as if this disaccharide werea classical enzyme-binding inhibitor. The possible mechanismsunderlying the nonlinearity of the trehalose-mediated inhibitionare discussed below.

Temperature dependence of the trehalose effect on the H⁺-ATPase. Trehalose accumulation in the yeast cytoplasm during heat shock(37 to 40°C) results in enhanced survival, apparently throughthe protection of protein structure against denaturation (18).On the other hand, enzyme catalysis is sensitive to temperature(42), and so it was decided to determine the effect of temperatureon the kinetics of the H⁺-ATPase in the absence and presenceof trehalose (Fig. 2). Increasing the temperature of assay resultedin an exponential increase in V_max (Fig. 2 and Table 1) andin a decrease in enzyme cooperativity, while the S_0.5 was onlyslightly modified, suggesting that the affinity of the enzymefor ATP is not sensitive to temperature (Table 1). As a resultof the increase in V_max and the lack of modifications in S_0.5,the catalytic efficiency increased with temperature (Table 1).In regard to the trehalose effect on enzyme activity, it wasobserved that, as the reaction temperature increased, the effectof trehalose on V_max gradually decreased, becoming negligibleat 40°C (Table 1). In contrast, the trehalose-induced increasein S_0.5 was constant at all temperatures tested. Thus, the estimatedcatalytic efficiency V_max/S_0.5 exhibited a trehalose-dependentdecrease (Table 1). The combined effects of temperature at 40°C(heat shock temperature) and trehalose (0.6 and 0.8 M) resultedin a calculated V_max/S_0.5 which was similar to that estimatedin the absence of the disaccharide at 25°C and only slightlylower than that observed at 30°C (Table 1). Trehalose didnot affect the cooperative behavior of the enzyme at any ofthe temperatures tested (Table 1). Therefore, at high temperatures,the H⁺-ATPase is only marginally inhibited by trehalose andits activity is comparable to that obtained when the enzymeis assayed in the absence of trehalose at the optimal temperaturefor yeast growth of 30°C (17).

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FIG. 2. Temperature effects on the kinetics of the plasma membrane H⁺-ATPase. Experimental conditions were as described for Fig. 1. The assay temperatures were 20°C ( ${circ}$ ), 25°C (•), 30°C ( ${square}$ ), 35°C ( ${blacksquare}$ ), and 40°C ( ${triangleup}$ ). The points are the means of three independent experiments. The standard deviations were less than 5% of the values. The lines are the result of the best fit to the Hill equation by nonlinear regression (equation 2).

Effect of trehalose on the energy of activation for ATP hydrolysis. The observed trehalose-induced decrease in V_max suggested thattrehalose increases the energy of activation (E_a) for the plasmamembrane H⁺-ATPase-catalyzed reaction. To further analyze this,E_a was determined at different trehalose concentrations by meansof the Arrhenius equation (Fig. 3). In the absence of trehalose,E_a was equal to 70.2 ± 0.9 kJ mol^-1. This value was similarto that reported elsewhere for the S. cerevisiae enzyme (33),suggesting that the two enzymes have similar mechanisms of catalysis.The presence of increasing concentrations of trehalose resultedin an increase in the energy barrier for ATP hydrolysis, asindicated by the rise in E_a as a function of trehalose concentration(Fig. 3, inset), e.g., in the presence of 0.8 M trehalose, E_aincreased to 89.0 ± 2.2 kJ mol^-1. Such an increase seemedto be exponential (Fig. 3, inset), probably as a result of theexponential increase in solvent viscosity induced by trehalose(see below).

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FIG. 3. Arrhenius plot for the plasma membrane H⁺-ATPase in the presence of different concentrations of trehalose. The points are the calculated V_max at each trehalose concentration and temperature. Trehalose molar concentrations were 0 ( ${circ}$ ), 0.2 (•), 0.4 ( ${square}$ ), 0.5 ( ${blacksquare}$ ), 0.6 ( ${triangleup}$ ), and 0.8 ( ${blacktriangleup}$ ). The lines were obtained by linear regression. (Inset) Plot of the energy of activation (E_a) versus trehalose concentration. E_a values were calculated from the line slopes as described by the Arrhenius equation (equation 3), and the line was the result of the fitting as described for Fig. 1C.

Effect of trehalose and temperature on viscosity. Kramer's theory for reactions in solution, which is applicableto protein folding and enzyme kinetics, states that solutionviscosity has a negative effect on the substrate probabilityof reaching the activated state and proceeding toward the product(21, 22), i.e., increasing solvent viscosity should result inlower protein conformational motility and lower reaction rates.In this regard, it has been reported elsewhere that solventviscosity is affected by changes in carbohydrate concentrationand temperature (26, 32). Thus, we began by testing the effectof increasing trehalose concentrations on viscosity at differenttemperatures (Fig. 4). At each temperature tested, increasingtrehalose concentrations increased exponentially the viscosityof the solution ( ${eta}$ ) (Fig. 4), e.g., at 20°C ${eta}$ increased from0.98 ± 0.03 cP in the absence of trehalose to 2.58 ±0.10 cP in the presence of 0.8 M trehalose (Fig. 4). In addition,increasing temperatures from 20 to 40°C resulted in a decreasein the trehalose-promoted viscosity, e.g., at 0.5 M trehalose, ${eta}$ decreased from 1.81 ± 0.04 cP at 20°C to 1.04 ±0.03 cP at 40°C (a viscosity value similar to that of waterat 25°C) (Fig. 4).

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FIG. 4. Effects of temperature on the viscosity of trehalose solutions. Each trehalose solution was prepared, poured into the viscometer, and degassed. Then the viscometer was immersed in a water jacket connected to a circulating water bath (Polyscience 9000) at the indicated temperature. Temperature in the viscometer was allowed to equilibrate 10 min before the falling times were read. The reaction mixture contained 10 mM PIPES, pH 7.0, and the following molar concentrations of trehalose: 0 ( ${circ}$ ), 0.2 (•), 0.4 ( ${square}$ ), 0.5 ( ${blacksquare}$ ), 0.6 ( ${triangleup}$ ), and 0.8 ( ${blacktriangleup}$ ).

Correlation between viscosity and H⁺-ATPase inhibition. In order to address the possible mechanism underlying the nonlinearinhibition of the H⁺-ATPase by increasing trehalose concentrations(Fig. 1C), as well as the lack of V_max inhibition by increasingtemperature (Table 1), the data were analyzed as described byJacob and Schmid (21) by using Kramer's theory (21, 22). Ifa reaction obeys Kramer's theory, a linear relationship shouldbe observed when the rate constant of the reaction is plottedagainst solution viscosity (20, 21). Thus, the relative V_maxvalues (V_max0/V_max) were plotted against the relative viscosityvalues ( ${eta}$ / ${eta}$ ₀) at each temperature tested (Fig. 5), and a lineardependence was observed as predicted (21, 22). Thus, the trehalose-mediatedinhibition of the H⁺-ATPase is most likely caused by the increasein the viscosity of the solution. In addition, although trehalosestill was able to increase solvent viscosity at 40°C (Fig.4), it seems that, at this temperature, the enzyme gains sufficientenergy to overcome the frictional effect and, as a result, structuralmovements involved in catalysis are not inhibited.

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FIG. 5. Effects of medium viscosity on the V_max displayed by the H⁺-ATPase. Experiments were performed at different temperatures. The relative V_max (V_max0/V_max) was plotted against the relative viscosity ( ${eta}$ / ${eta}$ ₀). ${eta}$ ₀ is the viscosity of the solution in the absence of trehalose, and V_max0 is the V_max in the absence of trehalose. Temperatures were 20°C ( ${circ}$ ), 25°C (•), 30°C ( ${square}$ ), 35°C ( ${blacksquare}$ ), and 40°C ( ${triangleup}$ ). The lines are the result of linear regressions of the data.

H⁺-ATPase inhibition by other viscosity-generating solutes. In addition to trehalose, sucrose and glycerol were used totest the generality of the correlation between increased viscosityand inhibition of the H⁺-ATPase. Both of these compounds arewidely used to preserve molecule function and/or structure duringhandling or storage. At 20°C, sucrose displayed the sameinhibition pattern as trehalose (data not shown). In contrast,higher glycerol concentrations were needed to inhibit the H⁺-ATPase(1 to 3.5 M), although the enzyme was inhibited to a higherextent than in the presence of trehalose (Fig. 6). In solutionscontaining increasing concentrations of sucrose or glycerol,it was observed that viscosities were similar to those obtainedfor trehalose when they were referred to solute mass fractionas described by the Mooney equation (equation 4) (Fig. 7A).Both sucrose and glycerol inhibited the H⁺-ATPase through adecrease in V_max. Furthermore, plotting the relative V_max0/V_maxagainst the relative viscosity ( ${eta}$ / ${eta}$ ₀) resulted in the linear relation(Fig. 7B) predicted by Kramer's equation (equation 5). For comparison,the data for trehalose were also included in Fig. 7B, and itmay be observed that sucrose and trehalose displayed the samerelation between V_max and ${eta}$ , while glycerol produced higher inhibitionof V_max as ${eta}$ increased (Fig. 7B). The pattern of enzyme inhibitioncorrelated with the solution viscosity increase as predictedby Kramer's theory (21, 22) and indicating that enzyme domainimmobilization was probably involved in inhibition.

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FIG. 6. Glycerol-mediated inhibition of the plasma membrane H⁺-ATPase. Experimental conditions were as described for Fig. 1. Glycerol molar concentrations were 0 ( ${circ}$ ), 1 (•), 2 ( ${square}$ ), 3 ( ${blacksquare}$ ), and 3.5 ( ${triangleup}$ ). The lines are the result of fitting the data to the Hill equation (equation 2) by nonlinear regression.

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FIG. 7. Correlation between viscosity and H⁺-ATPase inhibition by trehalose, sucrose, or glycerol. Viscosity measurements were performed as described for Fig. 4 at 20°C. Enzyme activity was measured as described for Fig. 1. Solutes were trehalose ( ${circ}$ ), sucrose (•), or glycerol ( ${square}$ ). (A) Plot of viscosity ( ${eta}$ ) versus mass fraction (c). The solid lines were the result of fitting the data to the Mooney equation (equation 4) by nonlinear regression. (B) Plot of the relative V_max (V_max0/V_max) against the relative viscosity ( ${eta}$ / ${eta}$ ₀) in the presence of different viscosity-generating agents. The lines are linear regressions of the data.

DISCUSSION

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Discussion

The H⁺-ATPase from K. lactis, like other P-ATPases, possessesthree cytoplasmic domains that undergo major conformationalchanges while alternating between two widely different states,E₁ and E₂ (29), as the enzyme alternatively binds substrates,hydrolyzes ATP, and pumps protons (29, 33, 41). Trehalose inhibitedthe H⁺-ATPase through a decrease in V_max and in V_max/S_0.5 (Table1). Since trehalose does not bind to the enzyme, the observedupward, nonlinear inhibition cannot be accounted for by thesame mechanisms involved in parabolic inhibition caused by anenzyme-binding inhibitor (Fig. 1C).

Kramer's theory predicts that solvent viscosity inhibits conformationalchanges in proteins and that this results in inhibition of enzymaticcatalysis (22). Indeed, the trehalose-, sucrose-, or glycerol-mediatedinhibition of V_max increased linearly with the viscosity ofthe solution (Fig. 5 and 7B). When the effect of trehalose wastested at increasing temperatures, inhibition of V_max decreased,and this was related to the decrease in viscosity. In addition,as temperature provided the system with higher energy, frictionalrestriction was overcome and as a result protein conformationalchanges involved in catalysis were facilitated. Furthermore,our data suggest that inhibition resulted from an increase inthe activation energy for ATP hydrolysis, i.e., the energy barrierseparating the two conformational states (E₁ and E₂) increased(Fig. 3) (33). In studies of the H⁺-ATPase from Schizosaccharomycespombe, Felix et al. (11) concluded that, at high temperatures,trehalose protects the structure of the enzyme by stabilizingstate E₂. Thus, it would be safe to assume that, in the H⁺-ATPasefrom S. pombe, the transition from a trehalose-stabilized E₂state to the E₁ conformation would require higher energy. Inaddition, it was recently reported elsewhere that, in the coldshock protein (CspB) folding reaction, the diffusion of proteinchain segments relative to the solvent is required to crossthe activated state, and this folding reaction displays an inverse,linear dependence on solvent viscosity (20), again in agreementwith Kramer's theory (22). Our data confirm the proposal that,in enzymes where a major conformational change is involved incatalysis, this change becomes the rate-limiting step in highlyviscous solutions (30). In contrast, as temperature increases,viscosity decreases and conformation interconversion is facilitated;in this case, the chemical reaction may become the rate-limitingstep (30).

The large increase in S_0.5 mainly observed at high trehaloseconcentrations (0.6 and 0.8 M) may be due to inhibition of thediffusion of the substrate (ATP) across the trehalose mesh surroundingthe protein (32). In agreement with our results, it has beenreported elsewhere that high concentrations of sorbitol anddextran inhibit the diffusion of ATP away from yeast mitochondria(1).

The estimated catalytic efficiency (V_max/S_0.5) of the H⁺-ATPasewas similar at 40°C in the presence of 0.8 M trehalose andat 25°C in the absence of trehalose (Table 1). Thus, atthe high temperatures where trehalose protects the H⁺-ATPasefrom denaturation (34), enzyme activity is near normal values,i.e., at heat shock temperatures trehalose would be a compatiblesolute (40). In contrast, trehalose-mediated inhibition of theH⁺-ATPase becomes important at temperatures where no enzymeprotection is needed, i.e., 20 to 30°C. In this regard,it has been reported elsewhere that, in the cytoplasm of yeast,viscosity is twice as high as water (44). Such viscosity doesallow for the rotational motion of some enzymes such as hexokinaseand phosphoglycerate kinase, while motional restriction of others,such as pyruvate kinase, is observed (44). Thus, viscosity shouldbe taken into consideration when the application of enzyme kineticsstudies to the in situ situation is attempted.

It may be hypothesized that, in vivo, the synthesis and accumulationof trehalose have to be finely regulated depending on environmentalconditions: (i) during heat shock, yeast cells accumulate trehalosein order to protect enzymes from denaturation while allowingfor the high rates of catalysis needed to survive stress (5),and (ii) as soon as the heat shock condition is over, yeastcells hydrolyze trehalose, thus allowing viscosity-sensitiveenzymes to display full activity (this report) and facilitatingdestabilization and refolding of those proteins that were partiallydenatured during the stress period (39).

The plasma membrane H⁺-ATPase from K. lactis exhibited sigmoidalkinetics, with a mean Hill number of 1.68 at 20°C, i.e.,this enzyme should be added to the growing number of E₁E₂-ATPaseswhere cooperativity has been detected (10, 29). The cooperativityof diverse H⁺-ATPases has led to the proposal that the functionalenzyme is probably not a monomer, but a dimer or even a hexamer(29).

The trehalose concentration at which H⁺-ATPase inhibition wasobserved was similar to that reported elsewhere to protect theenzyme against high temperatures (34) or against lyophilization(35). Thus, it is suggested that both the protection and theinhibition are due to the same trehalose-protein interactionmechanism. This mechanism would account for both the advantageof accumulating cytoplasmic trehalose during stress and theneed to hydrolyze this disaccharide immediately after the stressperiod is over (18, 43). Understanding the mechanism by whichtrehalose protects macromolecules against heat and desiccationmay lead to the design of conditions under which trehalose maybe applied to enzyme-dependent industrial processes.

ACKNOWLEDGMENTS

This work was partially funded by grants from CONACYT, 27568-N,and from DGAPA-UNAM IN207600. J.G.S. is a CONACYT Ph.D. fellow.

Rodrigo A. Díaz-Ruiz performed the viscosity measurements.Ramón Méndez and Norma Silvia Sánchez areacknowledged for their technical assistance.

FOOTNOTES

* Corresponding author. Mailing address: Departamento de Bioquímica, Instituto de Fisiología Celular, Universidad Nacional Autónoma de México (UNAM), A.P. 70-242, 04510 Mexico City, DF, México. Phone: (5255)-5622-5632. Fax: (5255)-5622-5630. E-mail: jsamped{at}ifisiol.unam.mx.

REFERENCES

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