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.

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.

Control of activity states of heart mitochondrial ATPase. Role of the proton-motive force and Ca2+.

The ATPase complex of submitochondrial particles exhibits activity transitions that are controlled by the natural ATPase inhibitor (Gómez-Puyou, A., Tuena de Gómez-Puyou, M. and Ernster, L. (1979) Biochim. Biophys. Acta 547, 252-257). The ATPase of intact heart mitochondria also shows reversible activity transitions; the activation reaction is induced by the establishment of electrochemical gradients, whilst the inactivation reaction is driven by collapse of the gradient. In addition it has been observed that the influx of Ca2+ into the mitochondria induces a rapid inactivation of the ATPase; this could be due to the transient collapse of the membrane potential in addition to a favorable effect of Ca2+-ATP on the association of the ATPase inhibitor peptide to F1-ATPase. This action of Ca2+ may explain why mitochondria utilize respiratory energy for the transport of Ca2+ in preference to phosphorylation. It is concluded that the mitochondrial ATPase inhibitor protein may exert a fundamental regulatory function in the utilization of electrochemical gradients.

Inactive to active transitions of the mitochondrial ATPase complex as controlled by the ATPase inhibitor.

The hydrolytic and phosphorylation activities of the ATPase complex of bovine heart mitochondria are regulated by the ATPase inhibitor of Pullman and Monroy [1]. The inhibiting action of the peptide on ATPase activity can be overcome by a proton-motive force. Submitochondrial particles that contain the inhibitor, either intrinsically or externally added, show a lag that precedes phosphorylation. Particles devoid of the inhibitor, of particles that are in an 'active' state fail to present the lag. Accordingly, the data indicate that, prior to the onset of phosphorylation, the ATPase complex undergoes a transition to an active state through a process that involves the inhibitor. The transition depends on the concentration of ATP, 50 microM ATP giving 50% inhibition of the proton-motive force-induced transition.

Extraction of mitochondrial membrane proteins into organic solvents in a functional state.

Protein-lipid complexes in organic solvents can be used as the starting material in the reassembly of functional planar and spherical bilayers (Montal, M., Darszon, A. and Schindler, H. (1981) Q. Rev. Biophys. 14, 1-79). The transfer of three enzymes of the inner mitochondrial membrane into organic solvents as protein-lipid complexes has been studied to understand better the extraction process. The enzymes studied were cytochrome c oxidase, ATPase and succinate dehydrogenase. These enzymes were transferred into hexane and diethyl ether in an active state, however, the activities extracted varied quantitatively, depending on the amount of protein of the starting preparation, the concentration of phospholipids and the cation employed. In all conditions cytochrome c oxidase was extracted with the highest yield and specific activity, and it was actually enriched in the organic extract. The values for succinate dehydrogenase and ATPase were lower, but their specific activities were similar to those of the starting material. This indicates that some membrane proteins are preferentially extracted into organic solvents in a functional state. The enzymes, as protein-lipid complexes, are fairly stable in organic solvents; in a month of storage at 4 degrees C in hexane some enzymes loose less than 50% of their activity.

The interaction of mitochondrial F1-ATPase with the natural ATPase inhibitor protein.

The interaction of soluble mitochondrial ATPase from beef heart with the natural ATPase inhibitor was studied. It was found that the phosphorylation of small amounts of ADP by phosphoenolpyruvate and pyruvate kinase, and an ensuing catalytic cycle supports the binding of the inhibitor to the enzyme. The association of the inhibitor with F1-ATPase does not increase the content of ATP in the F1-ATPase-inhibitor complex. The inhibitor of catalytic activity bathophenanthroline-Fe2+ chelate prevents the interaction, while the association of the inhibitor with F1-ATPase is delayed if the reaction is carried out in 2H2O. The date indicate that a transient state involved in the catalytic cycle is the form of the enzyme that interacts with the inhibitor. The proton-motive force-induced dissociation of the inhibitor from particulate ATPase is prevented by bathophenanthroline-Fe2+ chelate and nitrobenzofurazan chloride, which indicates that a functional catalytic (beta) subunit is required for the proton-motive force-induced release of the inhibitor. The data suggest a direct involvement of catalytic (beta) subunit in the mechanism by which the F1-ATPase senses the proton-motive force.

Action of alkyl cations and the natural ATPase inhibitor from mitochondria on soluble mitochondrial ATPase.

The effect of the natural ATPase inhibitor and octylguanidine on the ATPase activity of soluble oligomycin-insensitive mitochondrial F1 were compared. Both compounds induced a maximal inhibition of 60-80% in various preparation of F1 studied. The inhibition was of the uncompetitive type with respect to MgATP, and the action of the compounds was partially additive. The data suggest that octylguanidine reproduces the action of the natural ATPase inhibitor. Alkylammonium salts also affect the ATPase activity in a similar form. F1 bound to Sepharose-hexylammonium is largely inactive, whilst free hexylammonium at higher concentrations induces only a partial inhibition of the activity. This suggests that the degree of immobilization of F1 is related to the magnitude of inhibition of ATPase activity induced by alkyl cations. The binding of F1 to Sepharose-hexylammonium is prevented by high concentrations of Na+ or K+.

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.

Using evolutionary changes to achieve species-specific inhibition of enzyme action--studies with triosephosphate isomerase.

BACKGROUND: Many studies that attempt to design species-specific drugs focus on differences in the three-dimensional structures of homologous enzymes. The structures of homologous enzymes are generally well conserved especially at the active site, but the amino-acid sequences are often very different. We reasoned that if a non-conserved amino acid is fundamental to the function or stability of an enzyme from one particular species, one should be able to inhibit only the enzyme from that species by using an inhibitor targeted to that residue. We set out to test this hypothesis in a model system. RESULTS: We first identified a non-conserved amino acid (Cys14) whose integrity is important for catalysis in triosephosphate isomerase (TIM) from Trypanosoma brucei. The equivalent residues in rabbit and yeast TIM are Met and Leu, respectively. A Cys14Leu mutant of trypanosomal TIM had a tendency to aggregate, reduced stability and altered kinetics. To model the effects of a molecule targeted to Cys14, we used methyl methanethiosulfonate (MMTS) to derivatize Cys14 to a methyl sulfide. This treatment dramatically inhibited TIMs with a Cys residue at a position equivalent to Cys14, but not rabbit TIM (20% inhibition) or yeast TIM (negligible inhibition), which lack this residue. CONCLUSIONS: Cys14 of trypanosomal TIM is a non-conserved amino acid whose alteration leads to loss of enzyme structure and function. TIMs that have a cysteine residue at position 14 could be selectively inhibited by MMTS. This approach may offer an alternative route to species-specific enzyme inhibition.

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.

Thermostability of membrane systems in organic solvents.

Two multisubunit enzymes of the inner mitochondrial membrane, cytochrome oxidase and the H+-ATPase may be transferred into highly apolar solvents as protein-lipid complexes. At 70 degrees C and an initial water concentration of 13 microliters per ml organic solvent (toluene), the half-life of the ATPase was approx. 11 h, whereas that of cytochrome oxidase was about 100 s. Thermostability of cytochrome oxidase could be increased more than 100-times by decreasing the water concentration to 3 microliters per ml toluene. At this latter concentration of water the half-life of the ATPase at 90, 80 and 70 degrees C was 5, 48 and 96 h, respectively.

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.

Properties and regulation of the H+-ATP synthase of mitochondria.

A brief survey is made of the function of the H+-ATP synthase of mitochondria with emphasis on how it is regulated. A main regulatory factor is a low molecular weight protein whose binding to the enzyme appears to be essential for optimal accumulation of ATP as driven by electron transport. The ATP synthase is also controlled by ADP that, by binding to a site in the enzyme, inhibits ATP hydrolysis. Data on the spontaneous synthesis of a tightly bound ATP are discussed. Apparently, this requires proper subunit interactions to yield a competent catalytic site.

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.

Extraction of mitochondrial protein-lipid complexes into organic solvents: an approach to study the interaction between the ATPase and the mitochondrial ATPase-inhibitor protein.

Protein-lipid complexes were transferred directly from mitochondria and submitochondrial particles into hexane and ether. The protein-lipid residue left after solvent removal from these extracts was used to form liposomes which display low-temperature-resistant ATPase activity. Centrifugation experiments indicate that the ATPase activity is associated to the vesicles. Most of the F1-ATPases appear to be accessible to the external water phase of the liposomes. The ATPase activity of these particles was insensitive to dicyclohexylcarbodiimide and oligomycin. Incubation of these vesicles at room temperature activated (4--10-fold) the ATPase through a process that is partially sensitive to phenylmethylsulfonyl fluoride. The results with purified ATPase-inhibitor protein and (F1--ATPase)-inhibitor complex indicate that the activation process in the liposomes is due to the abolition of the inhibitory action of the inhibitor protein bound to a large fraction of the extracted ATPases. Liposomes prepared from hexane extracts obtained from submitochondrial particles having different levels of ATPase activity displayed an activation ratio which correlated with the number of ATPases that are inhibited by the inhibitor protein in the submitochondrial particles. The extraction of mitochondrial ATPase and its incorporation into liposomes followed by activity measurements may be used to judge the number of ATPases that in a given preparation contain the inhibitor protein in its inhibiting site.

Characteristics of the movement of K+ across the mitochondrial membrane and the inhibitory action of Tl+.

The incubation of mitochondria in mixtures that contain phosphate, NaCl, oxidizable substrate, and ethylenediaminetetraacetate induces the efflux of K-+. This process depends on electron transport and on the cyclic movement of phosphate across the membrane. Sodium ions, Li-+, or Cs-+ to a smaller extent, are required for maximal release of K-+. Potassium ions do not induce net efflux of internal K-+, but instead prevent the Na-+-induced release of K-+. Significant K-+ influx takes place in K-+-depleted mitochondria through a process with characteristics which are almost identical with those in which K-+ release takes place. As Na-+ inhibits the uptake of K-+, it is suggested that the movement of K-+ across the membrane is controlled by the cationic environment. Thallous ion, at concentrations that do not affect oxidative phosphorylation, was found to be an effective inhibitor of the influx and the efflux of K-+. The inhibitory effect of Tl-+ seems to be specific for K-+ since it does not affect the movement of Na-+. Mitochondria bind 10 to 15 nmol of 204-Tl-+ per mg of protein through an energy-independent process.

On the function of the natural ATPase inhibitor protein in intact mitochondria.

The recently described methodology to extract the mitochondrial ATPase along with other mitochondrial proteins into organic solvents, and their subsequent incorporation into liposomes [Eur. J. Biochem. (1982) 121, 427-433] has been employed to estimate the number of ATPases that contain the natural ATPase inhibitor protein in its inhibitory site in intact mitochondria incubated in various metabolic states. It was found that in the presence of electrochemical gradients about 50% of the ATPases are without inhibitor protein in its inhibitory site (active ATPases). In the transition from state 4 to state 3 the percentage of active ATPases diminishes from about 50% to approximately 20%. This indicates that during steady-state phosphorylation only a limited number of ATPases are in the active catalytic state, and that not only during active hydrolysis does the inhibitor protein interact with its inhibiting site; rather the inhibitor seems to interact with an intermediate state of the enzyme that appears either during the synthetic or hydrolytic reactions. In addition it was found that ATP, with or without uncoupler, induces the interaction of the inhibitor protein in more than 80% the ATPases through an oligomycin-sensitive process. Thus, notwithstanding other factors the interaction may account for the low hydrolytic activity of mitochondria.