Caenorhabditis elegans ATPase inhibitor factor 1 (IF1) MAI-2 preserves the mitochondrial membrane potential (Δψm) and is important to induce germ cell apoptosis.

When the electrochemical proton gradient is disrupted in the mitochondria, IF1 (Inhibitor Factor-1) inhibits the reverse hydrolytic activity of the F1Fo-ATP synthase, thereby allowing cells to conserve ATP at the expense of losing the mitochondrial membrane potential (Δψm). The function of IF1 has been studied mainly in different cell lines, but these studies have generated contrasting results, which have not been helpful to understand the real role of this protein in a whole organism. In this work, we studied IF1 function in Caenorhabditis elegans to understand IF1´s role in vivo. C. elegans has two inhibitor proteins of the F1Fo-ATPase, MAI-1 and MAI-2. To determine their protein localization in C. elegans, we generated translational reporters and found that MAI-2 is expressed ubiquitously in the mitochondria; conversely, MAI-1 was found in the cytoplasm and nuclei of certain tissues. By CRISPR/Cas9 genome editing, we generated mai-2 mutant alleles. Here, we showed that mai-2 mutant animals have normal progeny, embryonic development and lifespan. Contrasting with the results previously obtained in cell lines, we found no evident defects in the mitochondrial network, dimer/monomer ATP synthase ratio, ATP concentration or respiration. Our results suggest that some of the roles previously attributed to IF1 in cell lines could not reflect the function of this protein in a whole organism and could be attributed to specific cell lines or methods used to silence, knockout or overexpress this protein. However, we did observe that animals lacking IF1 had an enhanced Δψm and lower physiological germ cell apoptosis. Importantly, we found that mai-2 mutant animals must be under stress to observe the role of IF1. Accordingly, we observed that mai-2 mutant animals were more sensitive to heat shock, oxidative stress and electron transport chain blockade. Furthermore, we observed that IF1 is important to induce germ cell apoptosis under certain types of stress. Here, we propose that MAI-2 might play a role in apoptosis by regulating Δψm. Additionally, we suggest that IF1 function is mainly observed under stress and that, under physiological conditions, this protein does not play an essential role.

Equilibrium between monomeric and dimeric mitochondrial F1-inhibitor protein complexes.

Mg-ATP particles from bovine heart mitochondria have more than 95% of their F1 in complex with the inhibitor protein (IF1). The F1-IF1 complex was solubilized and purified. The question addressed was if this naturally occurring complex existed as monomers or dimers. Size exclusion chromatography and electron microscopy showed that most of the purified F1-IF1 complex was a dimer of two F1-IF1. As determined by the former method, the relative concentrations of dimeric and monomeric F1-IF1 depended on the concentration of protein that was applied to the column. Apparently, there is an equilibrium between the two forms of F1-IF1.

Dimethylsulfoxide promotes K+-independent activity of pyruvate kinase and the acquisition of the active catalytic conformation.

Pyruvate kinase requires K+ for maximal activity; the enzyme exhibits 0.02% of maximal activity in its absence [Kayne, F. J. (1971) Arch. Biochem. Biophys. 143, 232-239]. However, pyruvate kinase entrapped in reverse micelles exhibits an important K+-independent activity [Ramírez-Silva, L., Tuena de Gómez-Puyou, M., & Gómez-Puyou, A. (1993) Biochemistry 32, 5332-5338]. It is possible that the amount of water, as well as interactions of the protein with the micelles, can account for this behavior. We therefore explored the solvent effects on the catalytic properties of muscle pyruvate kinase. The enzyme exhibited an activity of 19.4 micromol x min(-1) x mg(-1) in 40% dimethylsulfoxide, compared with 280 and 0.023 micromol x min(1) x mg(-1) observed with and without K+ in water, respectively. pH activity profiles and kinetic constants for the substrates of pyruvate kinase in dimethylsulfoxide without K+ were similar to those in 100% water with K+, and differed from those in water without K+. The spectral center of mass of the emission spectrum of pyruvate kinase in 100% water exhibited a blue shift of 3.5 nm in the presence of Mg(2+), phosphenolpyruvate, and K+, ligands that induce the active conformation of the enzyme. The spectral center of mass of the apoenzyme in 30-40% dimethylsulfoxide coincided with that of the enzyme-Mg(2+)-phosphenolpyruvate-K+ complex in 100% water. The water relaxation rate enhancement factor and binding of phosphenolpyruvate to the pyruvate kinase-Mn(2+)-(CH3)4N+ complex in 30-40% dimethylsulfoxide were similar to those of the pyruvate kinase-Mn(2+)-K+ complex in water. The aforementioned results indicate that when muscle pyruvate kinase is without K+, 30-40% dimethylsulfoxide induces its active conformation.

Structural alterations and inhibition of unisite and multisite ATP hydrolysis in soluble mitochondrial F1 by guanidinium chloride.

The effect of guanidinium chloride (GdnHCl) on the ATPase activity and structure of soluble mitochondrial F1 was studied. At high ATP concentrations, hydrolysis is carried by the three catalytic sites of F1; this reaction was strongly inhibited by GdnHCl concentrations of <50 mM. With substoichiometric ATP concentrations, hydrolysis is catalyzed exclusively by the site with the highest affinity. Under these conditions, ATP binding and hydrolysis took place with GdnHCl concentrations of >100 mM; albeit at the latter concentration, the rate of hydrolysis of bound ATP was lower. Similar results were obtained with urea, although nearly 10-fold higher concentrations were required to inhibit multisite hydrolysis. GdnHCl inhibited multisite ATPase activity by diminishing the V(max) of the reaction without significant alterations of the Km for MgATP. GdnHCl prevented the effect of excess ATP on hydrolysis of ATP that was already bound to the high-affinity catalytic site. With and without 100 mM GdnHCl and 100 microM [3H]ATP in the medium, F1 bound 1.6 and 2 adenine nucleotides per F1, respectively. The effect of GdnHCl on some structural features of F1 was also examined. GdnHCl at concentrations that inhibit multisite ATP hydrolysis did not affect the exposure of the cysteines of F1, nor its intrinsic fluorescence. With 100 mM GdnHCl, a concentration at which unisite ATP hydrolysis was still observed, 0.7 cysteine per F1 became solvent-exposed and small changes in its intrinsic fluorescence of F1 were detected. GdnHCl concentrations on the order of 500 mM were required to induce important decreases in intrinsic fluorescence. These changes accompanied inhibition of unisite ATP hydrolysis. The overall data indicate that increasing concentrations of GdnHCl bring about distinct and sequential alterations in the function and structure of F1. With respect to the function of F1, the results show that at low GdnHCl concentrations, only the high-affinity site expresses catalytic activity, and that inhibition of multisite catalysis is due to alterations in the transmission of events between catalytic sites.

Synthesis and hydrolysis of ATP and the phosphate-ATP exchange reaction in soluble mitochondrial F1 in the presence of dimethylsulfoxide.

In medium containing 40% dimethylsulfoxide, soluble F1 catalyzes the hydrolysis of ATP introduced at concentrations lower than that of the enzyme [Al-Shawi, M.K. & Senior, A.E. (1992), Biochemistry 31, 886-891]. At this concentration of dimethylsulfoxide, soluble F1 also catalyzes the spontaneous synthesis of a tightly bound ATP to a level of approximately 0.15 mol per mol F1 [Gómez-Puyou, A., Tuena de Gómez-Puyou, M. & de Meis, L. (1986) Eur. J. Biochem. 159, 133-140]. The mechanisms that allow soluble F1 to carry out these apparently opposing reactions were studied. The rate of hydrolysis of ATP bound to F1 under uni-site conditions and that of synthesis of ATP were markedly similar, indicating that the two ATP molecules lie in equivalent high affinity catalytic sites. The number of enzyme molecules that have ATP at the high affinity catalytic site under conditions of synthesis or uni-site hydrolysis is less than the total number of enzyme molecules. Therefore, it was hypothesized that when the enzyme was treated with dimethylsulfoxide, a fraction of the F1 population carried out synthesis and another hydrolysis. Indeed, measurements of the two reactions under identical conditions showed that different fractions of the F1 population carried out simultaneously synthesis and hydrolysis of ATP. The reactions continued until an equilibrium level between F1.ADP + Pi <--> F1.ATP was established. At equilibrium, about 15% of the enzyme population was in the form F1.ATP. The DeltaG degrees of the reaction with 0.54 microM F1, 2 mM Pi and 10 mM Mg2+ at pH 6.8 was -2.7 kcal.mol-1 in favor of F1.ATP. The DeltaG degrees of the reaction did not exhibit important variations with Pi concentration; thus, the reaction was in thermodynamic equilibrium. In contrast, DeltaG degrees became significantly less negative as the concentration of dimethylsulfoxide was decreased. In water, the reaction was far to the left. The equilibrium constant of the reaction diminished linearly with an increase in water activity. The effect of solvent is fully reversible. In comparison to other enzymes, F1 seems unique in that solvent controls the equilibrium that exists within an enzyme population. This results from the effect of solvent on the partition of Pi between the catalytic site and the medium, and the large energetic barrier that prevents release of ATP from the catalytic site. In the presence of dimethylsulfoxide and Pi, ATP is continuously hydrolyzed and synthesized with formation and uptake of Pi from the medium. This process is essentially an exchange reaction analogous to the phosphate-ATP exchange reaction that is catalyzed by the ATP synthase in coupled energy transducing membranes.

Synthesis and release of ATP by soluble mitochondrial F1 in complex with its inhibitor protein during dimethylsulfoxide-water transitions.

Soluble mitochondrial F1 and F1 in complex with the natural ATPase inhibitor protein (F1-IP) catalyze the spontaneous synthesis of [gamma-32P]ATP from medium [32P]phosphate and enzyme-bound ADP when incubated in media with dimethylsulfoxide (Me2SO); under these conditions, the synthesized [gamma-32P]ATP is not released into the media, it remains tightly bound to the enzymes [Gómez-Puyou, A., Tuena de Gómez-Puyou, M. & de Meis, L. (1986) Eur. J. Biochem. 159, 133-140]. Some of the characteristics of the synthesized [gamma-32P]ATP were studied in F1 and F1-IP (ATPase activities of 70 and 1-3 micromol x min(-1) x mg(-1), respectively). In Me2SO media, gamma-phosphate of synthesized ATP in F1 or F1-IP exchanges with medium phosphate. From the rates of the exchange reaction, the half-times for hydrolysis of the synthesized ATP in F1 and F1-IP were calculated: 45 min and 58 min for F1 and F1-IP, respectively. The course that synthesized [gamma-32P]ATP follows after dilution of the Me2SO synthetic mixture with aqueous buffer was determined. After dilution, the half-life of synthesized ATP in F1 was less than 1 min. In F1-IP, ATP was also hydrolyzed, but at significantly lower rates. In F1-IP, dilution also produced release of the synthesized [gamma-32P]ATP. This was assayed by the accessibility of [gamma-32P]ATP to hexokinase. About 25% of [gamma-32P]ATP synthesized in F1-IP, but not in F1, was released into the media after dilution with aqueous buffer that contained 20 mM phosphate. Release of tightly bound ATP required the binding energy of phosphate and solvation of F1-IP, however, the particular kinetics of F1-IP were also central for medium ATP synthesis in the absence of electrochemical H+ gradients.

Sulfhydryl reagent susceptibility in proteins with high sequence similarity--triosephosphate isomerase from Trypanosoma brucei, Trypanosoma cruzi and Leishmania mexicana.

The amino acid sequence of triosephosphate isomerase from Trypanosoma brucei, Trypanosoma cruzi, and Leishmania mexicana have an identity of 68%. Using the numbering system for the T. brucei enzyme, in their aligned sequences, the T. cruzi and leishmanial enzymes have cysteine residues at positions 14, 40, 117 and 126. T. brucei triosephosphate isomerase has cysteine residues at positions 14, 40 and 126, and a valine residue at position 117. Dithionitrobenzoic acid and methylmethane thiosulfonate inhibited the three enzymes, but T. cruzi triosephosphate isomerase was more than 100-fold more sensitive. The sensitivity of wild type triosephosphate isomerase from T. cruzi and T. brucei to the reagents was equal to that of the Cys117Val and Val117Cys mutant enzymes, respectively. Triosephosphate isomerases that have cysteine residues at positions 40 and 126, but lack a cysteine residue at position 14 are insensitive to methylmethane thiosulfonate. Thus, sulfhydryl reagents act on Cys14. At stoichiometric concentrations, the reagents inhibited the three enzymes as a consequence of structural alterations as measured by binding of 8-anilino-1-napthalenesulfonic acid to previously buried hydrophobic regions. However, the times for half-maximal alterations were 10 min, 15 hours and over 30 hours for T. cruzi, T. brucei and L. mexicana triosephosphate isomerase, respectively. The effect of pH on the action of the sulfhydryl reagents and molecular modeling showed no differences in the solvent accessibility of Cys14. As Cys14 forms part of the dimer interface, the data indicate that, in the three enzymes, barriers of different magnitude hinder the interaction between the sulfhydryl reagents and Cys14. The barrier is lower in T. cruzi triosephosphate isomerase which makes its dimer interface more susceptible for perturbation.

Enzymes in low water systems.

Water is fundamental for enzyme action and for formation of the three-dimensional structure of proteins. Hence, it may be assumed that studies on the interplay between water and enzymes can yield insight into enzyme function and formation. This has proven correct, because the numerous studies that have been made on the behavior of water-soluble and membrane enzymes in systems with a low water content (reverse micelles or enzymes suspended in nonpolar organic solvents) have revealed properties of enzymes that are not easily appreciated in aqueous solutions. In the low water systems, it has been possible to probe the relation between solvent and enzyme kinetics, as well as some of the factors that affect enzyme thermostability and catalysis. Furthermore, the studies show that low water environments can be used to stabilize conformers that exhibit unsuspected catalytic properties, as well as intermediates of enzyme function and formation that in aqueous media have relatively short life-times. The structure of enzymes in these unnatural conditions is actively being explored.

Acceleration of unisite catalysis of mitochondrial F1-adenosinetriphosphatase by ATP, ADP and pyrophosphate--hydrolysis and release of the previously bound [gamma-32P]ATP.

The effect of ATP, ADP and pyrophosphate (PPi) on hydrolysis and release of [gamma-32P]ATP bound to the high-affinity catalytic site of soluble F1 from bovine heart mitochondria under unisite conditions [Grubmeyer, C., Cross, R. L. & Penefsky, H. S. (1982) J. Biol. Chem. 257, 12092-12100] was studied. In accord with the previous data, it was observed that millimolar concentrations of ATP or ADP added to F1 undergoing unisite hydrolysis of [gamma-32P]ATP accelerated its hydrolysis. PPi also produced a hydrolytic burst of a fraction of the previously bound [gamma-32P]ATP; kinetic data suggested that for production of optimal hydrolysis by PPi of the bound [gamma-32P]ATP, two binding sites with apparent Kd of 27 microM and 240 microM must be filled. The extent of the hydrolytic burst induced by MgPPi was lower than that induced by ADP and ATP. In F1 in which PPi had produced a hydrolytic burst of the bound [gamma-32P]ATP, the addition of ATP induced a second burst of hydrolysis. By filtration experiments and enzyme trapping, it was also studied whether ATP, ADP and PPi produce release of the tightly bound [gamma-32P]ATP. At millimolar concentrations, ATP and ADP brought about release of about 25% of the previously bound [gamma-32P]ATP. At micromolar concentrations, ADP accelerated the hydrolysis of the previously bound [gamma-32P]ATP but not its release. Hence, the hydrolytic and release reactions could be separated, indicating that the two reactions require the occupancy of different sites in F1. With PPi, no release of the tightly bound [gamma-32P]ATP was observed. The ADP induced hydrolysis and release of the F1-bound [gamma-32P]ATP were inhibited by sodium azide to the same extent (60%). Since release of ATP from a high-affinity catalytic site of F1 represents the terminal step of oxidative phosphorylation, the data illustrate that the binding energy of substrates to F1 is critical to the ejection of ATP into the media. The failure of PPi to induce release of [gamma-32P]ATP bound to F1 under unisite conditions is probably due to its lower binding energy.

Cloning, expression, purification and characterization of triosephosphate isomerase from Trypanosoma cruzi.

The gene that encodes for triosephosphate isomerase from Trypanosoma cruzi was cloned and sequenced. In T. cruzi, there is only one gene for triosephosphate isomerase. The enzyme has an identity of 72% and 68% with triosephosphate isomerase from Trypanosoma brucei and Leishmania mexicana, respectively. The active site residues are conserved: out of the 32 residues that conform the interface of dimeric triosephosphate isomerase from T. brucei, 29 are conserved in the T. cruzi enzyme. The enzyme was expressed in Escherichia coli and purified to homogeneity. Data from electrophoretic analysis under denaturing techniques and filtration techniques showed that triosephosphate isomerase from T. cruzi is a homodimer. Some of its structural and kinetic features were determined and compared to those of the purified enzymes from T. brucei and L. mexicana. Its circular dichroism spectrum was almost identical to that of triosephosphate isomerase from T. brucei. Its kinetic properties and pH optima were similar to those of T. brucei and L. mexicana, although the latter exhibited a higher Vmax with glyceraldehyde 3-phosphate as substrate. The sensitivity of the three enzymes to the sulfhydryl reagent methylmethane thiosulfonate (MeSO2-SMe) was determined; the sensitivity of the T. cruzi enzyme was about 40 times and 200 times higher than that of the enzymes from T. brucei and L. mexicana, respectively. Triosephosphate isomerase from T. cruzi and L. mexicana have the three cysteine residues that exist in the T. brucei enzyme (positions 14, 39, 126, using the numbering of the T. brucei enzyme); however, they also have an additional residue (position 117). These data suggest that regardless of the high identity of the three trypanosomatid enzymes, there are structural differences in the disposition of their cysteine residues that account for their different sensitivity to the sulfhydryl reagent. The disposition of the cysteine in triosephosphate isomerase from T. cruzi appears to make it unique for inhibition by modification of its cysteine.

Isolation and comparative studies of mitochondrial F1-ATPase from rat testis and beef heart.

The isolation and properties of F1-mitochondrial ATPase from rat testis are described. The isolation medium involves a chloroform extraction, and it is suitable even with small amounts of starting material that have a relatively low specific activity as in the case of rat testis submitochondrial particles. The isolated enzyme from rat testis had a specific activity of 30-45 mumol Pi/min/mg protein, which could be increased up to 90 mumol Pi/min/mg protein only in the presence of bicarbonate and maleate. The isolated enzyme represented less than 0.6% of the initial membrane proteins. It exhibited a typical five-band pattern in sodium dodecyl sulfate gel electrophoresis. However, it showed a ratio of subunits alpha:beta higher than the heart enzyme; its significance is unknown. The purified enzyme was cold labile and inhibited by natural ATPase inhibitor protein from bovine heart mitochondria and by dicyclohexylcarbodiimide. The results presented suggest that the low ATPase activity of testis submitochondrial particles is due to a reduced content of the F1-ATPase.

The contribution of water to the selectivity of pyruvate kinase for Na+ and K+.

For many years it has been known that K+ is an essential activator of pyruvate kinase [Kachmar, J. F. & Boyer, P. D. (1953) J. Biol. Chem. 200, 669-683] and that Na+ induces relatively small enhancements of activity. The effect of these two alkali metal ions on the activity of pyruvate kinase entrapped in the low water environment of reverse 'micelles formed with cetyltrimethylammonium, hexanol, n-octane and various water concentrations was studied. In reverse micelles with 3.6% water, the activity with 7 mM Na+ is more than 82 times higher than in aqueous solution with an equivalent Na+ concentration. As the concentration of water in reverse micelles is raised, the activating effect of relatively low concentrations of Na+ (or K+) decreases simultaneously to a more than 100-fold increase in the concentration of Na+ or K+ required for attaining half-maximal activation. Similar results were obtained with NH4+, Rb+ and Cs+. Therefore, the amount of water in the system is critical for observing activation by alkali metal ions. In fact, the concentration of Na+ required for half-maximal activation in standard aqueous media is higher than the concentrations that can be experimentally assayed. As evidenced from fluorescence and kinetic data, it appears that the entrapment of pyruvate kinase in reverse micelles does not produce gross structural alterations. Therefore, it is suggested that in conventional aqueous systems, the basis of the high discrimination between Na+ and K+ by pyruvate kinase is the higher energy required for desolvating Na+. Nevertheless, at all the water concentrations studied, the activities reached with K+ were higher than with Na+ which suggests that the Na+ form of the enzyme has a lower catalytic capacity than the K+-enzyme complex.

Synthesis of medium pyrophosphate by soluble mitochondrial F1 through dimethyl sulfoxide-water transitions.

Soluble F1 from heart mitochondria incubated in mixtures that have Mg2+, inorganic phosphate, and dimethyl sulfoxide (40% (v/v)) catalyzes the spontaneous synthesis of ATP and pyrophosphate (Tuena de Gómez-Puyou, M., García, J. J., and Gómez-Puyou, A. (1993) Biochemistry 32, 2213-2218). By filtration techniques, it was determined that synthesized ATP and pyrophosphate are enzyme bound, albeit the affinity for pyrophosphate was lower than that of ATP. After ATP and pyrophosphate were formed in dimethyl sulfoxide mixtures, dilution with aqueous buffer to a dimethyl sulfoxide concentration of 6.0% brought about the partition of pyrophosphate into the media. This was evidenced by filtration experiments as well as by the accessibility of synthesized pyrophosphate to soluble inorganic pyrophosphatase. Release of pyrophosphate induced by dilution occurred in less than 15 s. Under conditions that produce release of pyrophosphate, no release of ATP was observed; instead, ATP underwent hydrolysis. Studies on the effect of arsenate on the synthesis and hydrolysis of ATP and PPi in F1 showed that hydrolysis of synthesized PPi at its site of synthesis was slower than that of ATP. Thus, the question of whether differences in the rates of hydrolysis accounted for the dilution-induced release of PPi but not of ATP was addressed. Synthesis and hydrolysis of ATP and pyrophosphate were examined in preparations of soluble F1 in complex with its inhibitor protein; the complex had an ATPase activity about 100 times lower than that of free F1. In mixtures that contained dimethyl sulfoxide, the complex synthesized ATP and pyrophosphate at nearly the same rates; upon dilution, hydrolysis of both compounds occurred also at similar rates, yet only pyrophosphate was released. The same phenomenon was observed in F1 that had been depleted of adenine nucleotides. Hence, dilution-induced release of PPi was independent of the overall catalytic properties of the enzyme or its content of adenine nucleotides. Since synthesis of ATP occurs at the expense of the ADP that remains after depletion of adenine nucleotides, it is likely that the failure of ATP to be released is due to the high affinity that F1 exhibits for the synthesized ATP. Nevertheless, the results illustrate that a complete catalytic cycle that starts with medium Pi and ends with medium pyrophosphate may be reproduced in soluble mitochondrial F1.

Inhibition by trifluoperazine of ATP synthesis and hydrolysis by particulate and soluble mitochondrial F1: competition with H2PO4-.

The effect of trifluoperazine (TFP) on the ATPase activity of soluble and particulate F1-ATPase and on ATP synthesis driven by succinate oxidation in submitochondrial particles from bovine heart was studied at pH 7.4 and 8.8. At the two pH, TFP inhibited ATP hydrolysis. Inorganic phosphate protected against the inhibiting action of TFP. The results on the effect of various concentrations of phosphate in the reversal of the action of TFP on hydrolysis at pH 7.4 and 8.8 showed that H2PO4- is the species that competes with TFP. The effect of TFP on oxidative phosphorylation was studied at concentrations that do not produce uncoupling or affect the aerobic oxidation of succinate (< 15 microM). TFP inhibited oxidative phosphorylation to a higher extent at pH 8.8 than at pH 7.4; this was through a diminution in the Vmax, and an increase in the Km for phosphate. Data on phosphate uptake during oxidative phosphorylation at several pH showed that H2PO4- is the true substrate for oxidative phosphorylation. Thus, in both synthesis and hydrolysis of ATP, TFP and H2PO4- interact with a common site. However, there is a difference in the sensitivity to TFP of ATP synthesis and hydrolysis; this is more noticeable at pH 8.8, i.e., ATPase activity of soluble F1 remains at about 40% of the activity of the control in a concentration range of TFP of 40-100 microM, whereas in oxidative phosphorylation 14 microM TFP produces a 60% inhibition of phosphate uptake.

Water requirements in monomer folding and dimerization of triosephosphate isomerase in reverse micelles. Intrinsic fluorescence of conformers related to reactivation.

The possibility of using reverse micelles to stabilize monomers prior to formation of dimeric triosephosphate isomerase (TPI) from rabbit muscle was studied. TPI denatured with guanidine hydrochloride undergoes reactivation in reverse micelles formed with n-octane, hexanol, cetyltrimethylammonium bromide, and water. Reactivation of around 80% is observed at TPI concentrations of about 2 micrograms/mL of reverse micelles and water concentrations above 4.0%. With 3.0% water, reactivation is about 10%. If denatured TPI is incubated for a few seconds in reverse micelles with 5.0% water (or higher) followed by incubation in 3.0% water, reactivation is between 35% and 50%. That is, a brief exposure of denatured TPI to reverse micelles with a relatively high water concentration yielded a significant amount of structures competent for formation of catalytically active dimers. As evidenced by kinetic data, these structures correspond to monomers of TPI [Garza-Ramos, G. Tuena de Gómez-Puyou, M., Gómez-Puyou, A., & Gracy R. W. (1992) Eur. J. Biochem. 208, 389-395]. After a 5-2.0% water transition, competent monomers were stabilized for at least 30 min; a subsequent rise in water concentration led to dimerization and appearance of activity. By changes in the amount of water, it was possible to determine in reverse micelles the amount of water required for monomer folding and dimerization; i.e., less water was required in the dimerization step. Experiments with a model system, trypsin and the soybean inhibitor, showed that, in reverse micelles with 2.0% water, protein-protein interactions readily take place.(ABSTRACT TRUNCATED AT 250 WORDS)

Deamidation of triosephosphate isomerase in reverse micelles: effects of water on catalysis and molecular wear and tear.

The specific deamidation of asparagine-71 of triosephosphate isomerase increases upon substrate binding and catalysis. This deamidation at the dimer interface initiates subunit dissociation, unfolding, and protein degradation. The apparent connection between catalysis and terminal marking supports the concept of "molecular wear and tear", and raises questions related to the molecular events that lead to deamidation. In order to explore this interaction, triosephosphate isomerase was entrapped in reverse micelles with different water contents that support different catalytic rates. Deamidation was quantified for the free enzyme, the enzyme in the presence of substrates, and the enzyme which had been covalently modified at the catalytic center with the substrate analogue 3-chloroacetol phosphate (CAP). Both in water and in reverse micelles of cetyltrimethylammonium with 3% and 6% water, substrate binding enhanced deamidation. Studies of the extent of deamidation at various water concentrations showed that deamidation per catalytic turnover was about 6 and 17 times higher in 6% and 3% water than in 100% water, respectively. The enzyme was also entrapped in micelles formed with toluene, phospholipids, and Triton X-100 to explore the process at much lower water concentrations (e.g., 0.3%). Under these conditions, catalysis was very low, and hardly any deamidation took place. Deamidation of the CAP-labeled enzyme was also markedly diminished. At these low-water conditions, the enzyme exhibited markedly increased thermostability and resistance to hydrolysis of the amide bonds. The data suggest that the rate of deamidation not only is dependent on the number of catalytic events but also is related to the time that asparagine-71 exists in a conformation or solvent environment more favorable for deamidation.

Activity and fluorescence changes of lactate dehydrogenase induced by guanidine hydrochloride in reverse micelles.

Denaturants activate several multimeric enzymes in reverse micelles [Garza-Ramos, G., Darszon, A., Tuena de Gómez-Puyou, M. & Gómez-Puyou, A. (1992) Eur. J. Biochem. 205, 509-517]. Here, the effect on activity and intrinsic fluorescence of pig heart lactate dehydrogenase (LDH) in reverse micelles [formed with 0.2 M cetyltrimethylammonium bromide in octane/hexanol (8.6:1, by vol.)] was explored at various water and guanidine hydrochloride (Gdn/HCl) concentrations. Emission fluorescence spectra of LDH in aqueous media and in micelles were similar. As in all aqueous media, 1.0 M Gdn/HCl in the water phase of reverse micelles produced fluorescence quenching and a blue shift of the maximal emission. In 5.0 M Gdn/HCl, instead of the red shift and significant quenching seen in water, the maximum emission further shifted to the blue and was only slightly quenched. Gdn/HCl titrations of activity and fluorescence changes of LDH in micelles with different water contents showed that at Wo ([H2O]/[surfactant]) of 6.6, 8.3, or 12.5, increasing concentrations of Gdn/HCl up to 0.6 M produced small changes in fluorescence, whereas activity increased several-fold. At higher denaturant concentrations, activity decreased with significant fluorescence changes. In reverse micelles with 1 M Gdn/HCl, Vmax but not Km of LDH decreased with time. Under these conditions, there was progressive quenching of LDH fluorescence. The results show that in reverse micelles different Gdn/HCl concentrations induce variations in activity with or without alterations of the intrinsic fluorescence of LDH. The results also indicate that in reverse micelles, concentrations of Gdn/HCl below 1.0 M cause an enhancement of protein flexibility; this is accompanied by a marked increase in activity without important changes in intrinsic fluorescence. 1.0 M Gdn/HCl produces perturbations of inter-subunit contacts that lead to fluorescence quenching and loss of catalytic activity, probably as consequence of dimerization of tetrameric LDH.

Synthesis of pyrophosphate and ATP by soluble mitochondrial F1.

Soluble F1 from beef heart that has been depleted of adenine nucleotides to values of 0.4 mol of ADP and 0.1 mol ATP/mol of enzyme has the capacity to synthetize about 0.1 mol of ATP/mol of enzyme from medium phosphate in the presence of Mg2+ and 30% dimethyl sulfoxide. Under the same conditions, native and adenine nucleotide depleted F1 can also synthesize pyrophosphate to values that range from 0.03 to 0.05 mol/mol of F1. The formation of pyrophosphate requires Mg2+ and dimethyl sulfoxide. The formed pyrophosphate remains bound to F1 during filtration through Sephadex centrifugation columns. In all water media, adenine nucleotide depleted, but not native, F1 can hydrolyze pyrophosphate to values of about 0.2 nmol min-1 mg-1. This activity is inhibited or stimulated by agents (adenylyl imidodiphosphate, aurovertin, and methanol) that produce such effects on the ATPase activity of F1; NaN3 stimulated the activity. Therefore, F1 from bovine heart mitochondria has the capacity to catalyze synthesis and hydrolysis of ATP. Synthesis of pyrophosphate by the soluble F1 appears to follow the same energetic considerations that have been postulated for ATP synthesis by the soluble enzyme [de Meis (1989) Biochim. Biophys. Acta 973, 339-349].

Dimerization and reactivation of triosephosphate isomerase in reverse micelles.

The reactivation of the homodimeric enzyme triosephosphate isomerase (TPI) was studied in reverse micelles. The enzyme was denatured in conventional aqueous mixtures with guanidine hydrochloride and transferred to reverse micelles formed with cetyltrimethylammonium bromide, hexanol, n-octane and water. In the transfer step, denatured TPI monomers distributed in single micelles, and guanidine hydrochloride was diluted more than 100 times. Under optimal reactivation conditions, 100% of the enzyme activity could be recovered. The rate of appearance of the catalytic activity increased with the concentration of protein, which indicated that catalysis required the formation of the dimer. The rate of TPI reactivation also increased with increasing protein concentration in the system with denatured TPI covalently derivatized at the catalytic site with the substrate analogue 3-chloroacetol phosphate. Thus, reactivation could take place via the formation of dimers composed of an inactive and an active subunit. Reactivation critically depended on the amount of water in the reverse micelles. The plot of the extent of reactivation versus the amount of water (2.5-7.0%) was markedly sigmoidal. Less than 20% reactivation took place with water concentrations below 3.5%, due to the formation (in less than 30 s) of stable inactive structures. The results indicate that reverse micelles provide a useful system to probe the events involved in the transformation of unfolded monomers to polymeric enzymes.

Activity of heart and muscle lactate dehydrogenases in all-aqueous systems and in organic solvents with low amounts of water. Effect of guanidine chloride.

The effect of urea and guanidine hydrochloride (GdmCl) on the activity of lactate dehydrogenases from heart and muscle was studied in standard water mixtures and in reverse micelles formed with n-octane, hexanol, cetyltrimethylammonium bromide and water in a concentration that ranged over 2.5-6.0% (by vol.). In all water mixtures GdmCl (0.15-0.75 M) and urea (0.5-3.0 M) inhibited the activity of the enzymes at non-saturating pyruvate concentrations. At concentrations of pyruvate that proved inhibitory for enzyme activity due to the formation of a ternary enzyme-NAD-pyruvate complex, GdmCl and urea increased the activity of the enzymes. This increase correlated with a decrease of the ternary complex, as evidenced by its absorbance at 320-325 nm. In the low-water system it was found that: (a) at all concentrations of pyruvate tested (0.74-30 mM), GdmCl enhanced the activity of the heart enzyme to a similar extent; (b) in the muscle enzyme, GdmCl inhibited or increased the activity through a process that depended on the concentration of pyruvate and GdmCl; (c) under optimal conditions, the activation by GdmCl was about two times lower in the muscle than in the heart enzyme, although in all-water media the activity of the muscle enzyme was twice as high. The expression of lactate dehydrogenase activity in the low-water system was higher with the heart than with the muscle enzyme compared to their activities in all-water media (about 260 and 600 mumol min-1 mg-1 in the heart and muscle enzymes respectively). Apparently for catalysis, the water requirement in the heart enzyme is lower than in the muscle enzyme. It is likely that the different response of the two enzymes to solvent is due to their distinct structural features.