A monoclonal antibody that inhibits Trypanosoma cruzi growth in vitro and its reaction with intracellular triosephosphate isomerase.

In parasites of the order Kinetoplastida, such as Trypanosoma cruzi and Trypanosoma brucei, glycolysis is carried out by glycolytic enzymes in glycosomes. One of the glycolytic enzymes is triosephosphate isomerase (TIM), which in T. brucei is localized exclusively in glycosomes, whereas in T. cruzi, the localization of TIM has not been fully ascertained. In the present work, we made a monoclonal antibody (mAb 6-11G) against recombinant T. cruzi TIM (rTcTIM). Incubation of T. cruzi epimastigotes with the mAb inhibited parasite survival. Western blotting showed that the mAb recognized rTcTIM and a 27 kDa band in T. cruzi lysates that corresponded to TcTIM. Sera from patients with Chagas disease recognized rTcTIM and cross-reacted with human recombinant TIM. The cross reactivity between parasite and human TIM possibly contributes to the autoimmune pathogenesis of Chagas disease. Electron microscopy of T. cruzi epimastigotes with the mAb showed that TIM was located within glycosomes, in the cytoplasm, the nucleus, and the kinetoplast. Collectively, the data shed new light on T. cruzi TIM and opens perspectives for drug design.

A hinge of the endogeneous ATP synthase inhibitor protein: the link between inhibitory and anchoring domains.

The ATP synthase of bovine heart mitochondria possesses a regulatory subunit called the endogenous inhibitory protein (IF(1)). This subunit regulates the catalytic activity of the F(1) sector in the mitochondrial inner membrane. When DeltamuH(+) falls, IF(1) binds to the enzyme and inhibits ATP hydrolysis. On the other hand, the establishment of a DeltamuH(+) induces the release of the inhibitory action of IF(1), allowing ATP synthesis to proceed. IF(1) is also involved in the dimerization of soluble F(1). Dynamic domain analysis and normal mode analysis of the reported crystallographic structure of IF(1) revealed that it has an effective hinge formed by residues 46-52. Molecular dynamics data of a 27 residue fragment confirmed the existence of the hinge. The hinge may act as a regulatory region that links the inhibitory and anchoring domains of IF(1). The residues assigned to the hinge are conserved between mammals, but not in other species, such as yeasts. Likewise, unlike the heart inhibitor, the yeast protein does not have the residues that allow it to form stable dimers through coiled-coil interactions. Collectively, the data suggest that the hinge and the dimerization domain of the inhibitor protein from bovine heart are related to its ability to form stable dimers and to interact with other subunits of the ATP synthase.

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.

Factors that control the reactivity of the interface cysteine of triosephosphate isomerase from Trypanosoma brucei and Trypanosoma cruzi.

The amino acid sequences and X-ray structures of homodimeric triosephosphate isomerase from the pathogenic parasites Trypanosoma brucei (TbTIM) and Trypanosoma cruzi (TcTIM) are markedly similar. In the two TIMs, the side chain of the only interface cysteine (Cys14) of one subunit docks into loop 3 of the other subunit. This portion of the interface is also markedly similar in the two enzymes. Nonetheless, Cys14 of TcTIM is nearly 2 orders of magnitude more susceptible to the thiol reagent methylmethane thiosulfonate (MMTS) than Cys14 of TbTIM. The causes of this difference were explored by measuring the second-order rate constant of inactivation by MMTS (k(2)) under various conditions. At pH 7.4, k(2) in TcTIM is 70 times higher than in TbTIM. The difference decreases to 30 when the amino acid sequence of loop 3 and adjoining residues of TbTIM are conferred to TcTIM (triple mutant). The pK(a) values of the thiol group of the interface cysteine of TcTIM and the triple mutant were 0.7 pH unit lower than in TbTIM. Because this difference could account for the different sensitivity of the enzymes to thiol reagents, we determined the k(2) of inactivation at equal levels of ionization of their interface cysteines. Under these conditions, the difference in k(2) between TcTIM and TbTIM became 8-fold, whereas that of the triple mutant to TbTIM was 1.5 times. The substrate analogue phosphoglycolate did not modify the pK(a) of the thiol group of the interface, albeit it diminished the rate of its derivatization by MMTS. In the presence of phosphoglycolate, under conditions in which the interface cysteines of the enzymes had equal levels of protonation, the difference in k(2) of TcTIM and TbTIM became smaller, whereas k(2) of the triple mutant was almost equal to that of TbTIM. Thus, from measurements of the reactivity of the interface cysteine in various conditions, it was possible to obtain information on the factors that control the dynamics of a portion of the dimer interface.

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.

Escherichia coli TEM1 beta-lactamase in CTAB reverse micelles: exchange/diffusion-limited catalysis.

We report kinetic data of penicillin hydrolysis catalyzed by beta-lactamase entrapped in reverse micelles formed with cetyl trimethylammonium bromide (CTAB), n-octane, hexanol and aqueous buffer. The K(cat) of this diffusion-limited reaction can be improved in aqueous buffer by a factor of 1.1-1.2 just by increasing the phosphate buffer concentration from 50 to 100 mM. In reverse micelles, increasing the buffer concentration has little effect on K(cat) when the size of the empty micelle is below the size of the protein. However, in larger micelles, the effect is enhanced and the K(cat) improves several fold, changing the form of the curve of K(cat) versus Wo from bell-shaped to almost hyperbolic. The results indicate that micellar exchange and internal diffusion may limit the reaction in reverse micelles and provide further evidence that the form of the curve depends on other factors besides the relationship between the size of the enzyme and that of the empty reverse micelle.

Crystal structure of triosephosphate isomerase from Trypanosoma cruzi in hexane.

To gain insight into the mechanisms of enzyme catalysis in organic solvents, the x-ray structure of some monomeric enzymes in organic solvents was determined. However, it remained to be explored whether the structure of oligomeric proteins is also amenable to such analysis. The field acquired new perspectives when it was proposed that the x-ray structure of enzymes in nonaqueous media could reveal binding sites for organic solvents that in principle could represent the starting point for drug design. Here, a crystal of the dimeric enzyme triosephosphate isomerase from the pathogenic parasite Trypanosoma cruzi was soaked and diffracted in hexane and its structure solved at 2-A resolution. Its overall structure and the dimer interface were not altered by hexane. However, there were differences in the orientation of the side chains of several amino acids, including that of the catalytic Glu-168 in one of the monomers. No hexane molecules were detected in the active site or in the dimer interface. However, three hexane molecules were identified on the surface of the protein at sites, which in the native crystal did not have water molecules. The number of water molecules in the hexane structure was higher than in the native crystal. Two hexanes localized at <4 A from residues that form the dimer interface; they were in close proximity to a site that has been considered a potential target for drug design.

Derivatization of the interface cysteine of triosephosphate isomerase from Trypanosoma brucei and Trypanosoma cruzi as probe of the interrelationship between the catalytic sites and the dimer interface.

In the interface of homodimeric triosephosphate isomerase from Trypanosoma brucei (TbTIM) and Trypanosoma cruzi (TcTIM), one cysteine of each monomer forms part of the intersubunit contacts. The relatively slow derivatization of these cysteines by sulfhydryl reagents induces progressive structural alterations and abolition of catalysis [Garza-Ramos et al. (1998) Eur. J. Biochem. 253, 684-691]. Derivatization of the interface cysteine by 5, 5-dithiobis(2-nitrobenzoate) (DTNB) and methylmethane thiosulfonate (MMTS) was used to probe if events at the catalytic site are transmitted to the dimer interface. It was found that enzymes in the active catalytic state are significantly less sensitive to the thiol reagents than in the resting state. Maximal protection against derivatization of the interface cysteine by thiol reagents was obtained at near-saturating substrate concentrations. Continuous recording of derivatization by DTNB showed that catalysis hinders the reaction of sulfhydryl reagents with the interface cysteine. Therefore, in addition to intrinsic structural barriers, catalysis imposes additional impediments to the action of thiol reagents on the interface cysteine. In TcTIM, the substrate analogue phosphoglycolate protected strongly against DTNB action, and to a lesser extent against MMTS action; in TbTIM, phosphoglycolate protected against the effect of DTNB, but not against the action of MMTS. This indicates that barriers of different magnitude to the reaction of thiol reagents with the interface cysteine are induced by the events at the catalytic site. Studies with a Cys14Ser mutant of TbTIM confirmed that all the described effects of sulfhydryl reagents on the trypanosomal enzymes are a consequence of derivatization of the interface cysteine.

Differences in the intersubunit contacts in triosephosphate isomerase from two closely related pathogenic trypanosomes.

The aligned amino acid sequences of TIM from Trypanosoma cruzi (TcTIM) and Trypanosoma brucei (TbTIM) have a positional identity of 68%. The two enzymes have markedly similar catalytic properties. Agents that interact with their interface Cys inhibit TcTIM and TbTIM; and those TIMs that lack this Cys (such as human TIM) are largely or completely insensitive to these agents. The susceptibility of TcTIM to the agents is approximately 100 times higher than that of TbTIM. To ascertain the cause of this large difference, the crystal structure of TcTIM was solved at 1.83 A resolution. The two enzymes are very similar homodimers. In TcTIM and TbTIM their respective Cys, 15 or 14, forms part of the dimer interface. In both, the contacts of the Cys with residues of the other subunit are almost identical. Nevertheless, there are noteworthy differences between the two; the existence of glutamine 18 in TbTIM instead of glutamic acid in TcTIM at the beginning of helix 1 decreases the contacts between this portion of the protein and helix 3 of the other subunit. In addition, TcTIM has proline at position 24 in the first helix of the TIM barrel; this is absent in the other TIM. Pro24 disrupts the regular helix arrangement, making the pitch of this helix 1.2 A longer than in TbTIM. When Pro24 of TcTIM was substituted for Glu, the sensitivity of TcTIM to sulfhydryl reagents increased about fivefold, possibly as a consequence of an increase in the space between the first portion of helix 1 and helix 3 of the other subunit. Therefore, it may be concluded that the geometry of the latter region is central in the accessibility to agents that perturb the interface Cys. In human TIM this region is more compact.

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.

Reactivation of triosephosphate isomerase from three trypanosomatids and human: effect of suramin.

The reactivation of the homodimeric triosephosphate isomerases (TIMs) from Trypanosoma brucei, T. cruzi, Leishmania mexicana and humans was determined after their denaturation with guanidine hydrochloride. In the range of 2-32 microg of T. brucei TIM per ml and 0.2-5 microg of the other enzymes per ml, the rate and extent of TIM reactivation depended on protein concentration, indicating that at these protein concentrations, the rate-limiting step of reactivation is monomer association and not monomer folding. The rate of monomer association was more than one order of magnitude lower in the T. brucei enzyme than in the other three enzymes. Suramin is a drug of choice in the treatment of sleeping sickness, but its mechanism of action is not known. At micromolar concentrations, Suramin inhibited the reactivation of the four enzymes, but the extent of inhibition by Suramin decreased with increasing protein concentration as consequence of a diminution of the life time of the folded monomer. Since the life time of the monomer of T. brucei TIM is longer than that of the other enzymes, Suramin is a more effective inhibitor of the reactivation of TIM from T. brucei, particularly at monomer concentrations above 1 microg of protein per ml (monomer concentration approx. 37 nM). Compounds that are structurally related to Suramin also inhibit TIM reactivation; their effect was about five times more pronounced in the enzyme from T. brucei than in human TIM.

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.

Unisite ATP hydrolysis by soluble Rhodospirillum rubrum F1-ATPase is accelerated by Ca2+

At saturating concentrations of ATP, soluble F1 from the Rhodospirillum rubrum (RF1) exhibits a higher rate of hydrolysis with Ca2+ than with Mg2+. The mechanisms involved in the expression of a higher catalytic activity with Ca2+ were explored by measuring the ATPase activity of RF1 at substiochiometric concentrations of ATP (unisite conditions). At a ratio of 0.25 [gamma-32P]ATP per RF1, the enzyme exhibited a 50 times higher hydrolytic rate with Ca2+ than with Mg2+. The rate of [gamma-32P]ATP binding to RF1 was in the same range with the two divalent metal ions. Centrifugation-filtration of RF1 exposed to substoichiometric [gamma-32P]ATP concentrations and Mg2+ through Sephadex columns yielded an enzyme that contained [gamma-32P]ATP and [32P]phosphate in a stoichiometry that was close to one. In the presence of Ca2+, the eluted enzyme did not contain [gamma-32P]ATP nor [32P]phosphate. This indicated that the rate of product release was faster with Ca2+ than with Mg2+. It was also observed that the ratio of multisite to unisite hydrolysis rates was of similar magnitude with both divalent cations. This suggests that they do not affect differently the cooperative mechanisms that may exist between catalytic sites. In consequence, the higher ATPase activity of RF1 in presence of Ca2+ strongly suggests that the retention time of products is decreased in the presence of this cation. Copyright 1998 Elsevier Science B.V.

Reverse Micelle Systems Composed of Water, Triton X-100, and Phospholipids in Organic Solvents

Catalysis, stability, and thermostability of yeast hexokinase were determined in the microenvironments of two organic solvent/Triton X-100/phospholipids systems. In the abscence of enzyme, phase diagrams showed two transparent/turbid transitions, and reverse micelles were only observed in the second region of transparency (T2), where particle size as a function of water content shows a minima (see previous paper in this issue). In the present work, enzyme activity was detected throughout the four regions of the phase diagrams of these systems. Catalysis increased with water content; nevertheless, the maximum activities that were reached in the toluene and propylbenzene systems were 30 and 1.6%, respectively, of the activity in all aqueous media. Because in the T2 region in the propylbenzene system, micelles are much smaller than in toluene (see preceding paper), it would appear that expression of catalysis depends on the size of the micelles. However, a comparison of the dimensions of hexokinase and those of reverse micelles in the T2 region, suggests that in this region, hexokinase entrapment increases the inner volume of the micelle. High enzyme thermostability was only observed in the first transparent region (T1) of the system that contained phospholipids. In this region, hexokinase induced the formation of reverse micelles from dispersed surfactant monomers. There is a striking similarity in the dimensions of hexokinase entrapped in reverse micelles as determined by dynamic light scattering measurements in the T1 region with those of hexokinase as obtained from X ray diffraction studies of the enzyme in a crystalline environment. This suggest that high thermostability, and low catalytic rates result from restrictions in mobility imposed by a low water environment. Copyright 1998 Academic Press. Copyright 1998Academic Press

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.

Unisite hydrolysis of [gamma 32 P]ATP by soluble mitochondrial F1-ATPase and its release by excess ADP and ATP. Effect of trifluoperazine.

Some of the characteristics of unisite hydrolysis of [gamma 32P]ATP as well as the changes that occur on the transition to multisite catalysis were further studied. It was found that a fraction of [gamma 32P]ATP bound at the catalytic sites of F1 under unisite conditions undergoes both hydrolysis and release induced by medium nucleotides upon addition of millimolar concentrations of ADP or ATP. The fraction of [gamma 32P]ATP that undergoes release is similar to the fraction that undergoes hydrolytic cleavage, indicating that the rates of the release and hydrolytic reactions of bound [gamma 32P]ATP are in the same range. As part of studies on the mechanisms through which trifluoperazine inhibits ATP hydrolysis, its effect on unisite hydrolysis of [gamma 32P]ATP was also studied. Trifluoperazine diminishes the rate of unisite hydrolysis by 30-40%. The inhibition is accompanied by a nearly tenfold increase in the ratio of [gamma 32P]ATP/32Pi bound at the catalytic site and a 50% diminution in the rate of 32Pi release from the enzyme into the media. Trifluoperazine also induces heterogeneity of the three catalytic sites of F1 in the sense that in a fraction of F1 molecules, the high-affinity catalytic site has a turnover rate lower than the other two. Trifluoperazine does not modify the release of previously bound [gamma 32P]ATP induced by medium nucleotides. The latter indicates that hindrances in the release of Pi do not necesarily accompany alterations in the release of ATP even though both species lie in the same site.