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Originally published In Press as doi:10.1074/jbc.M408866200 on August 20, 2004

J. Biol. Chem., Vol. 279, Issue 44, 45613-45617, October 29, 2004
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Heat of PPi Hydrolysis Varies Depending on the Enzyme Used

YEAST AND CORN VACUOLAR PYROPHOSPHATASE*

Wagner S. da-Silva{ddagger}, Flavio M. Bomfim§, Antonio Galina, and Leopoldo de Meis||

From the Departamento de Bioquímica Médica, Instituto de Ciências Biomédicas, Universidade Federal do Rio de Janeiro, Cidade Universitária, Rio de Janeiro RJ, 21941–590, Brasil

Received for publication, August 3, 2004 , and in revised form, August 17, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
With yeast-soluble inorganic pyrophosphatase, the heat released during PPi hydrolysis was –6.3 kcal/mol regardless of the KCl concentration in the medium. With the membrane-bound pyrophosphatase of corn vacuoles, the heat released varies between –23.5 and –7.5 kcal/mol depending on the KCl concentration in the medium and whether or not a H+ gradient is formed across the vacuole membranes. The data support the proposal that enzymes are able to handle the energy derived from phosphate compound hydrolysis in such a way as to determine the parcel that is used for work and the fraction that is converted into heat.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Evidence reported in the past 5 years indicates that enzymes are able to handle the energy derived from the hydrolysis of phosphate compounds in such a way as to determine the parcel that is used for work and the fraction that is converted into heat (1–12). The ability to modulate the conversion of energy into either heat or work varies depending on both the enzyme and the experimental conditions used. This was first observed with the sarco/endoplasmic reticulum Ca2+-ATPases (SERCA),1 a family of membrane-bound ATPases that are able to translocate Ca2+ ion across the membrane by using the chemical energy derived from ATP hydrolysis. With these enzymes it was found that the heat released during ATP hydrolysis may vary from 10 to 30 kcal/mol depending on the SERCA isoform used and on whether or not a Ca2+ gradient is formed across the membrane (1–10). Kinetic measurements indicate that the SERCA are able to hydrolyze ATP through two catalytic routes (4–6, 8, 13–15). In one of them hydrolysis is coupled with the translocation of Ca2+ through the membrane. In this case, a part of the chemical energy derived from ATP cleavage is used for Ca2+ transport, and a part is converted into heat. The second route is a shortcut of the transport cycle where the cleavage of ATP is completed in a step that precedes the translocation of Ca2+ through the membrane, and all the energy derived from ATP hydrolysis is converted into heat (4, 5, 8, 10, 13–15). Thus, during the course of the reaction, the amount of heat released varies depending on how much ATP is cleaved in each of these two routes.

Recently, Bianconi (11) found that the enthalpy for the reactions of yeast hexokinase varies depending on the isozymes used, and Szöke et al. (12) in an elegant theoretical analysis proposed that enzyme could adjust the reaction path by altering the reversible interchange of different forms of energies (chemical, electrical, and mechanical). In these views, the same reaction catalyzed by different enzymes would lead to the release of different amounts of heat. In the report by Szöke et al. (12), it was proposed that the amount of heat produced during PPi hydrolysis by the soluble and membrane-bound PPases should be markedly different. The soluble PPase would convert {Delta}G into heat, whereas for the membrane-bound PPases, less heat should be released because part of the energy derived from PPi hydrolysis would be captured and used by the enzyme to pump protons across the membrane. In this report we measure the heat release during PPi hydrolysis using yeast-soluble inorganic PPase and the membrane-bound PPase of corn vacuoles. We found that indeed the amount of heat released during PPi hydrolysis varies depending on the enzyme used, but contrary to the proposal by Szöke et al. (12), the amount of heat released by vacuolar PPase was larger than that measured with yeast-soluble PPase.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Vacuolar Vesicles—Maize seeds were prepared from maize root cells (Zea mays L.) as previously described (16–18). Protein concentrations were determined by the method of Lowry et al. (19).

Proton Gradient—The accumulation of H+ by the vesicles was determined by measuring the fluorescence quenching of 9-amino-6-chloro-2-methoxyacridine (Molecular Probes) by using a spectrofluorimeter (model F-3010, Hitachi, Tokyo, Japan). The excitation wavelength was set at 415 nm, and the emission wavelength was set at 485 nm. The reaction was carried out in 2 ml of medium containing 10 mM MOPS-Tris, pH 7.5, 3 µM 9-amino-6-chloro-2-methoxyacridine, 1 mM MgCl2, 0.3 mM PPi, and different KCl concentrations. FCCP was dissolved in ethanol. The amounts used were such that the ethanol concentration in the reaction mixture was never higher than 0.5% (v/v). At this concentration, ethanol had no effect on either the H+ gradient or PPi hydrolysis. Yeast inorganic PPase was lyophilized from Saccharomyces cerevisiae (Sigma catalog number I-1643, lot 74H 7045). PPase activity was measured colorimetrically by determining the rate of Pi liberation (20).

Heat of Reaction—This was measured using an OMEGA Isothermal Titration Calorimeter from Microcal, Inc. (Northampton, MA). The calorimeter sample cell (1.5 ml) was filled with reaction medium, and the reference cell was filled with Milli-Q water. After equilibration at 30 °C, the reaction was started by injecting either soluble yeast PPase or vacuolar vesicles into the sample cell, and the heat change was recorded for 30 min. The heat change measured during the initial 2 min after injection of the enzymes was discarded in order to avoid artifacts such as heat derived from the dilution of the enzyme in the reaction medium and binding of ions to the enzymes. The duration of these events is less than 1 min (1, 7–10). Calorimetric enthalpy ({Delta}Hcal) is calculated by dividing the amount of heat released by the amount of PPi hydrolyzed. The units used are moles for substrate hydrolyzed and kilocalories for heat released. Negative values indicate that the reaction is exothermic, and positive values indicate that it is endothermic. The enthalpy of buffer protonation ({Delta}Hp) was measured at 30 °C by measuring the heat released following the addition of known amounts of HCl to the assay medium, and the value found was –3.8 kcal/mol (9).

Experimental Procedure—All experiments were performed at 30 °C. In a typical experiment, the assay media were divided in two samples, which were used for the simultaneous measurement of PPi hydrolysis and heat release. The syringe of the calorimeter was filled with either a solution of yeast PPase or a suspension of corn vacuoles, and the temperature difference between the syringe and the reaction cell of the calorimeter was allowed to equilibrate, a process that usually took between 8 and 12 min. During equilibration, the enzymes used for measurements of PPi hydrolysis were kept at the same temperature, length of time, and protein dilution as the enzymes kept in the calorimeter syringe. These different measurements were started simultaneously with enzymes to a final concentration of either 40.0 µg/ml for the corn vacuolar PPase and 0.2 µg/ml for yeast PPase.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Kinetics Properties—Plants, protozoa, and photosynthetic bacteria have a membrane-bound PPase, which is able to use the chemical energy derived from PPi cleavage to pump H+ across the membrane (16, 17, 21, 22). Potassium activates the hydrolysis of PPi, and the K+ concentration needed for half-maximal activation varies depending on the organism used to isolate the vacuoles (16, 17, 22–25). With the maize root vacuoles used in this work, half-maximal activation of PPi hydrolysis (Fig. 1A) and half-maximal proton gradient formation (Fig. 1B) were obtained in the presence of 5–10 mM KCl (Fig. 1A). Contrasting with the vacuolar PPase, the soluble yeast PPase does not convert chemical energy into osmotic energy and is not activated by K+. The optimum pH of both vacuolar and yeast PPase is in the range of pH 7.0–8.0 (data not shown). Accordingly, all our experiments were performed at pH 7.5. The yeast PPase is inhibited by NaF (26, 27), and in the conditions we used, half-maximal inhibition was attained in the presence of 0.12 mM NaF (Fig. 2). As for the various membrane-bound PPases so far described, the vacuolar PPase is not inhibited by NaF (22). In Fig. 2, concentrations of NaF higher than 1 mM were not used in order to avoid the formation of magnesium fluoride complexes and the decrease of the free Mg2+ concentration in the medium. The specific activity of the vacuolar PPase varies depending on the plant species and tissue used. The values previously reported for corn roots (16, 17, 22) vary between 0.06 and 0.09 µmol of PPi/mg·min–1. In this report, the activity among the various vacuoles preparations used varied between 0.05 and 0.11 µmol of PPi/mg·min–1. The specific activity of the purified soluble yeast PPase used was much higher than that of corn vacuoles and varied between 40 and 48 µmol of PPi/mg·min–1 (Fig. 1). In Refs. 22 and 28, the specific activity of vacuolar PPase purified from different plants varied between 3.0 and 14.2 µmol of PPi/mg·min–1.



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  		FIG. 1.
K+ dependence for PPase activity (A) and H+ transport (B). A, the assay medium composition was 50 mM MOPS-Tris buffer, pH 7.5, 0.3 mM PPi, 0.6 mM MgCl2, and different KCl concentrations as shown in the figure. {Delta}, yeast inorganic PPase; {circ}, corn vacuolar PPase. The values represent the mean ± S.E. of three experiments performed with three different vacuole preparations. B, the medium contained 10 mM MOPS-Tris, pH 7.0, 3 µM 9-amino-6-chloro-2-methoxyacridine, 0.3 mM PPi, 0.6 mM MgCl2, and either 100 mM KCl (line a), 25 mM KCl (line b),or without KCl addition (line c). Arrows indicate the addition of PPi and FCCP to final concentrations of 0.3 mM and 3 µM, respectively. The reaction was performed at 30 °C and was started by the addition PPase to a final concentration in the medium of 40.0 µg/ml for the maize root vacuoles and 0.2 µg/ml for yeast PPase.

 



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  		FIG. 2.
Effect of NaF on vacuolar ({circ}) and yeast () PPase. The assay medium contained 50 mM MOPS-Tris buffer, pH 7.5, 100 mM KCl, 0.3 mM PPi, and 0.6 mM MgCl2. Other conditions were as in Fig. 1A.

 
Heat Production during PPi Hydrolysis—Both with the vacuolar and the soluble yeast PPase the amount of heat released was proportional to the amount of PPi cleaved (Figs. 3 and 4). The {Delta}Hcal for PPi hydrolysis measured with yeast PPase was the same in the absence or in the presence of 100 mM KCl (Fig. 5 and Table I). The {Delta}Hcal values we found with yeast PPase are in the same range as those measured previously with yeast PPase by Ohlmeyer and Shatas (29) at {Delta}Hcal –8.95 kcal/mol and by Ging and Sturtevant (30) at {Delta}Hcal –5.81 kcal/mol.



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  		FIG. 3.
PPi hydrolysis and heat release by vacuolar PPase. The assay medium composition was 50 mM MOPS-Tris buffer, pH 7.5, 0.3 mM PPi, and 0.6 mM MgCl12 and either without added KCl () or with 100 mM KCl ({circ}). The reaction was performed at 30 °C and was started by the addition of PPase to a final protein concentration of 40.0 µg/ml. A, PPase activity; B, heat released; C, correlation between heat released and PPi hydrolyzed. The figure shows a typical experiment.

 



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  		FIG. 4.
PPi hydrolysis and heat release by yeast PPase. The assay medium composition was 50 mM MOPS-Tris buffer, pH 7.5, 0.3 mM PPi, 0.6 mM MgCl2, and 100 mM KCl. The reaction was performed at 30 °C and was started by the addition of PPase to a final protein concentration of 0.2 µg/ml PPase activity (A), heat released (B), and correlation between heat released and PPi hydrolyzed (C). The figure shows a typical experiment.

 



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  		FIG. 5.
Effect of K+ on the {Delta}Hcal of PPi hydrolysis. The experimental conditions were as described in Figs. 3 and 4 using inorganic PPase from yeast ({triangleup}) or vacuolar PPase in the absence ({circ}) or presence () of 5 µM FCCP. The figure shows a representative experiment.

 


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  		TABLE I
{Delta}Hcal values of yeast and corn vacuolar PPase

The experimental conditions and assay medium compositions were the same as those described in Figs. 3, 4, 5, 6. Negative {Delta}Hcal values indicate that the reaction is exothermic, and positive values indicate that it is endothermic. The differences between vacuolar and yeast PPase are statistically significant.

 
Most surprisingly, when the hydrolysis was catalyzed by the vacuolar PPase, the heat released during the cleavage of each PPi molecule was significantly larger than that measured with yeast PPase (Table I). As far as we know, the {Delta}Hcal for PPi hydrolysis has not been measured previously using a vacuolar PPase as catalyst. Different from the yeast PPase, with the vacuolar PPase the heat released during PPi varied depending on the K+ concentration in the medium (Figs. 3 and 5 and Table I). In the presence of a gradient and 5 mM KCl, the heat released was 22.8 kcal/mol PPi and decreased to 9.7 kcal/mol PPi as the K+ concentration in the medium was raised from 5 to 100 mM.

The proton ionophore FCCP impairs the formation of a H+ gradient (Fig. 1) and enhances the vacuolar PPase activity (Fig. 6), indicating that the accumulation of H+ in the vesicle lumen partially inhibits the hydrolytic activity of the enzyme. A similar phenomenon was previously observed with SERCA and is referred to as "back inhibition" (4). At all K+ concentrations tested, the heat released for each PPi molecule cleaved in the presence of a gradient was larger than that measured in the presence of FCCP (Fig. 6 and Table I).



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  		FIG. 6.
Effect of H+-gradient on the rates of PPi and heat release by corn vacuoles. The assay medium composition was 50 mM MOPS-Tris buffer, pH 7.5, 0.3 mM PPi, 0.6 mM MgCl2, and 5mM KCl in the presence () or absence ({circ}) of 5 µM FCCP. A, PPase activity; B, heat release; C, values of A and B were plotted as heat released versus PPi cleaved. The reactions were performed at 30 °C and started by the addition of corn vacuoles to a final concentration of 40 µg/ml. Values are mean ± S.E. of either 15 (without FCCP) or 8 (with FCCP) experiments.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The data presented show that the amount of heat released during PPi hydrolysis varies depending on the enzyme used. On the basis of the theoretical analysis by Szöke et al. (12), the finding that the vacuolar PPase released more heat per mol of PPi hydrolyzed than the soluble yeast PPase appears to be paradoxical. However, when compared with the SERCA (5–9), the data indicate that the vacuolar PPase can cleave PPi through two different catalytic routes with different caloric yields, and the choice between the two routes varies depending on the KCl concentration in the medium and on whether or not a H+ gradient is formed across the vesicle membranes.

Energy of Hydrolysis of Phosphate Compounds during Catalysis—A common feature of different enzymes involved in energy transduction is that during catalysis there is a significant decrease of the substrate energy level that takes place at the catalytic site before its cleavage (Table II). In these enzymes, work is performed during the decrease of the energy level of the phosphate compound. Data obtained in different laboratories indicate that the conversion of the phosphate compound from high to low energy is promoted by a change of water activity at the catalytic site of the enzyme, and this decrease can be explained by the solvation energy theory (33–39).


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  		TABLE II
Variability of the of the {Delta}G0 for the hydrolysis of phosphate compounds at the catalytic site of enzymes

The values shown in the table are from Refs. 33 and 39–45.

 
Ca2+ transport by SERCA involves phosphorylation by ATP of an aspartyl residue located at the catalytic site of the enzyme forming an acyl phosphate residue of high energy (Table II), and translocation of Ca2+ across the membrane occurs simultaneously with the conversion of the acyl phosphate from high into low energy (6, 13–15, 31–33). In leaky vesicles (no gradient) practically all the ATPase activity measured is coupled with the translocation of Ca2+ across the membrane. However, when intact vesicles are used, formation of a transmembrane gradient leads to a back inhibition of the ATPase activity and to a ramification of the catalytic cycle whereby the acyl phosphate of high energy is cleaved before its conversion into low energy. As a result, all the energy derived from the cleavage of the high energy acyl phosphate is converted into heat, and none is used for Ca2+ transport. Thus, the {Delta}Hcal for ATP hydrolysis varies between –10 and –12 kcal/mol in the absence of a gradient and between –22 and –30 in the presence of a Ca2+ gradient (for review see Ref. 6). Similar to SERCA 1, formation of H+ gradient in corn vacuoles leads a decrease of the PPase activity (Fig. 6) and to an increase of the amount of heat released during PPi hydrolysis (Figs. 5 and 6 and Table I).

PPi of High and Low Energy—Between 1979 and 1990 it was shown in different laboratories (40–43) that the soluble PPase catalyzed the rapid and reversible formation of enzyme-bound PPi from Pi (Fig. 7, step 3). The energy of the bound PPi is much smaller than that measured in an aqueous solution (Table II) (33, 37, 38, 44). In this same period, it was also shown that the energy of PPi hydrolysis could be drastically decreased ({Delta}G0 from –4.0 up to +2.0) by lowering the water activity of the medium with organic solvents (33, 37, 38, 45). Recently, it has been shown that the soluble PPase binds two Mg2+, and at the catalytic site each Mg2+ binds a water molecule. During catalysis the enzyme-substrate complex undergoes isomerization; one of the two water molecules bound to Mg2+ dissociates from the enzyme-substrate complex, and the second water molecule is used to hydrolyze PPi (26, 27, 46–48). Inhibition by NaF is promoted by replacement of the magnesium-bound water molecule by F– (26, 27, 47). According to the early measurements (40–43), conversion of PPi from high into low energy should occur during isomerization of the enzyme. Contrasting with the soluble PPase, little is know about the catalytic cycle of the vacuolar PPase. The fact that the vacuolar PPase is able to pump protons, is activated by K+ (Fig. 1), and is not inhibited by NaF (Fig. 2) indicates that there are significant differences between the catalytic cycles of the two PPases. The role of K+ on H+ transport and PPase activity is controversial (22). Patch clamp studies indicate that the vacuolar PPase uses the energy derived from PPi cleavage to translocate both H+ and K+ across the membrane (49–51), but this has not been confirmed in experiments with vacuolar PPase reconstituted into proteoliposomes (52, 53). Different from the soluble yeast PPase, the various vacuolar PPases so far described are not inhibited by F– (Fig. 2) (22), indicating that the hydrolysis of PPi at the catalytic site of the vacuolar PPase does not involve magnesium-bound water as determined in yeast PPase.



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  		FIG. 7.
Proposed reaction sequence for the vacuolar PPase. During step 2, protons are translocated across the vacuolar membrane, and simultaneously, there is a decrease of the hydrolysis energy of PPi bound at the catalytic site of the enzyme. In the sequence PPi refers to high energy pyrophosphate and PPi to low energy pyrophosphate. Step 4 would only be observed when a H+ gradient is formed across the vacuoles membrane.

 
In order to account for the large differences of {Delta}Hcal measured with the two PPases, we hypothesize the following. (i) Similar to the yeast PPase and to the various energy-transducing enzymes shown in Table II, the catalytic cycle of the vacuolar PPase would include two different enzyme-substrate complexes, one with high energy PPi and the second with low energy PPi ((Fig. 7, step 2). (ii) The translocation of H+ across the vacuolar membrane would be coupled with conversion of PPi from high into low energy at the catalytic site. (iii) Similar to SERCA 1, the vacuolar PPase would be able to catalyze the cleavage of PPi through two different routes, a slow one involving hydrolysis of high energy PPi (Fig. 7, step 4) and a faster route involving the conversion of PPi from high into low energy (Fig. 7, steps 2 and 3). The amount of heat released when the high energy PPi is cleaved (Fig. 7, step 4) would be significantly larger than that released when the low energy PPi is cleaved (Fig. 7, steps 2 and 3). (iv) The rate-limiting step of the catalytic cycle would be the conversion of the two enzyme-PPi complexes (Fig. 7, step 2) that would be regulated by K+ and by the H+ gradient. In the absence of K+, the enzyme form that binds high energy PPi would be accumulated, and most of the PPi hydrolyzed would be processed through step 4 in Fig. 7, which does not involve H+ transport. K+ would accelerate the conversion of PPi from high into low energy and both the rates of PPi hydrolysis and H+ transport (Fig. 1 and Fig. 7, steps 2 and 3). (v) Similar to SERCA 1, formation of a H+ gradient leads to a back inhibition (Fig. 6) that decreases the rate of conversion of PPi from high into low energy (step 2) and accelerates the hydrolysis of PPi through step 4. The formation of a H+ gradient and K+ would therefore have opposite effects on the rate of step 2 in Fig. 7.

In principle, the energy requirement of a reaction is not dependent on the mechanism of catalysis. The finding that the soluble yeast PPase produces less heat than the vacuolar PPase indicates that the soluble PPase yields a product that requires significant free energy input. This event probably takes place during the conversion of PPi from high into low energy. Although for the vacuolar enzyme H+ is translocated across the membrane during the transition, for the yeast PPase, the conversion of PPi from high into low energy is associated with the exclusion of water from the catalytic site.


   FOOTNOTES
 
* This work was supported in part by grants from PRONEX, Conselho Nacional de Desenvolvimento Científico e Tecnológico, and Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

{ddagger} Recipient of a fellowship from Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro. Back

§ Recipient of a fellowship from Conselho Nacional de Desenvolvimento Científico e Tecnológico, Brasil. Back

Research fellow of the Conselho Nacional de Desenvolvimento Científico e Tecnológico, Brasil. Back

|| To whom correspondence should be addressed. Tel./Fax: 55-21-2270-1635; E-mail: demeis{at}bioqmed.ufrj.br.

1 The abbreviations used are: SERCA, sarco/endoplasmic reticulum Ca2+-ATPases; FCCP, carbonyl cyanide p-trifluoromethoxyphenyl-hydrazone; MOPS, 4-morpholinepropanesulfonic acid; PPase, pyrophosphatase. Back


   ACKNOWLEDGMENTS
 
We are grateful to Valdecir A. Suzano and Antônio C. Miranda for technical assistance.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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