Studies on (Na+ + K+)-activated ATPase. XLIV. Single phosphate incorporation during dual phosphorylation by inorganic phosphate and adenosine triphosphate

$\text{(}\text{Na}{}^{\mathrm{+}}\text{+ K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ can be phosphorylated by its substrate ATP as well as by its product inorganic phosphate. The maximal capacity for phosphorylation by either of these two substances is one mol phosphate per mol enzyme. In order to investigate whether the enzyme molecule possesses only one phosphorylation site common to ATP and P_i, or two phosphorylation sites, one for ATP and one for P_i, dual phosphorylation of the enzyme has been carried out. Under conditions, which are maximally favourable for each type of phosphorylation, successive phosphorylation by P_i and ATP leads to a maximal incorporation of only one mol phosphate per mol enzyme. The phosphorylation capacity for ATP decreases by the same amount as the P_i-phosphorylation level increases, without an effect on the apparent affinity for ATP.The results can be explained by assuming either a single common phosphorylation site for P_i and ATP, or a conformational change of the enzyme following phosphorylation by P_i, which excludes phosphorylation by ATP.     

Adenylate kinase is a source of ATP for tumor mitochondrial hexokinase

It has proposed that hexokinase bound to mitochondria occupies a preferred site to wich ATP from oxidative phosphorylation is channeled directly (Bessman, S. (1966) Am. J. Medicine 40, 740–749). We have investigated this problem in isolated Zajdela hepatoma mitochondria. Addition of ADP to well-coupled mitochondria in the presence of an oxidizable substrate initiates the synthesis of glucose 6-phosphate via bound hexokinase. This reaction is only partially inhibited by oligomycin, carboxyatractyloside, carbonyl cyanide m-chlorophenylhydrazone (CCCP) ot any combination of these, suggesting a source of ATP in addition to oxidative phosphorylation. This source appears to be adenylate kinase, since Ado₂P₅, an inhibitor of the enzyme, suppresses hexokinase activity by about 50% when added alone or suppresses activity completely when added together with any of the inhibitors of oxidative phosphorylation. Ado₂P₅ does not uncouple oxidative phosphorylation nor does it inhibit ADP transport (state 3 respiration) or hexokinase. The relative amount of ATP contributed by adenylate kinase is dependent upon the ADP concentration. At low ADP concentraions, glucose phosphorylation is supported by oxidative phosphorylation, but as the adenine nucleotide translocator becomes saturated the ATP contributed by adenylate kinase increases due to the higher apparent K_m of the enzyme. Under conditions of our standard experiment ([ADP] = 0.5 mM), adenylate kinase provides about 50% of the ATP used by hexokinase in well-coupled mitochondria. In spite of this, externally added ATP supported higher rates of hexokinase activity than ADP. Our findings demonstrate that oxidative phosphorylation is not a specific or preferential source of ATP for hexokinase bound to hepatoma mitochondria. The apparent lack of a channeling mechanism for ATP to hexokinase in these mitochondria is discussed.     

多聚磷酸盐激酶双底物通道腔的理性设计及其在无细胞催化生产谷胱甘肽中的应用

三磷酸腺苷(adenosine triphosphate,ATP)是一种重要的辅助因子,参与许多需能的生物催化反应。多聚磷酸盐激酶(polyphosphate kinases,PPK)由于其底物聚磷酸盐廉价易得,可以为消耗ATP的反应提供能量。本研究选择哈氏噬纤维菌(Cytophaga hutchinsonii)来源的ChPPK,进行了底物谱和耐受性分析,通过分子对接和定点突变,理性改造多聚磷酸盐激酶的双底物通道腔来提高PPK酶的催化活性。与野生型相比,筛选得到突变体ChPPK_K81H-K103V的相对酶活提高了326.7%,同时,双突变扩大了ChPPK的底物利用范围与耐受性,提高了该酶的耐热性与耐碱性。基于该ATP再生系统,本研究偶联谷胱甘肽双功能酶GshAB和ChPPK_K81H-K103V,破细胞后采用无细胞催化生产谷胱甘肽,加入5 mmol/L ATP后,该体系6 h可以生产(25.4±1.9) mmol/L的谷胱甘肽,比突变前的催化体系提高了41.9%。优化无细胞催化体系的缓冲液、裂解液菌体量、补料时间后,无细胞体系可产生(45.2±1.8) mmol/L谷胱甘肽,底物l-半胱氨酸的转化率达到90.4%。提高ChPPK生产ATP的能力,可有效增强底物的转化率,降低催化成本,实现了无细胞催化生产谷胱甘肽的高产量、高转化率与高经济价值的统一。本研究提供了一种绿色高效的ATP再生系统,可为消耗ATP的生物催化反应平台提供可持续动力。     

The affinity purification and characterization of ATP synthase complexes from mitochondria

The mitochondrial F₁-ATPase inhibitor protein, IF₁, inhibits the hydrolytic, but not the synthetic activity of the F-ATP synthase, and requires the hydrolysis of ATP to form the inhibited complex. In this complex, the α-helical inhibitory region of the bound IF₁ occupies a deep cleft in one of the three catalytic interfaces of the enzyme. Its N-terminal region penetrates into the central aqueous cavity of the enzyme and interacts with the γ-subunit in the enzyme''s rotor. The intricacy of forming this complex and the binding mode of the inhibitor endow IF₁ with high specificity. This property has been exploited in the development of a highly selective affinity procedure for purifying the intact F-ATP synthase complex from mitochondria in a single chromatographic step by using inhibitor proteins with a C-terminal affinity tag. The inhibited complex was recovered with residues 1–60 of bovine IF₁ with a C-terminal green fluorescent protein followed by a His-tag, and the active enzyme with the same inhibitor with a C-terminal glutathione-S-transferase domain. The wide applicability of the procedure has been demonstrated by purifying the enzyme complex from bovine, ovine, porcine and yeast mitochondria. The subunit compositions of these complexes have been characterized. The catalytic properties of the bovine enzyme have been studied in detail. Its hydrolytic activity is sensitive to inhibition by oligomycin, and the enzyme is capable of synthesizing ATP in vesicles in which the proton-motive force is generated from light by bacteriorhodopsin. The coupled enzyme has been compared by limited trypsinolysis with uncoupled enzyme prepared by affinity chromatography. In the uncoupled enzyme, subunits of the enzyme''s stator are degraded more rapidly than in the coupled enzyme, indicating that uncoupling involves significant structural changes in the stator region.     

Loss of mitochondrial ATP synthase subunit beta (Atp2) alters mitochondrial and chloroplastic function and morphology in Chlamydomonas

Mitochondrial F₁F_O ATP synthase (Complex V) catalyses ATP synthesis from ADP and inorganic phosphate using the proton-motive force generated by the substrate-driven electron transfer chain. In this work, we investigated the impact of the loss of activity of the mitochondrial enzyme in a photosynthetic organism. In this purpose, we inactivated by RNA interference the expression of the ATP2 gene, coding for the catalytic subunit β, in the green alga Chlamydomonas reinhardtii. We demonstrate that in the absence of β subunit, complex V is not assembled, respiratory rate is decreased by half and ATP synthesis coupled to the respiratory activity is fully impaired. Lack of ATP synthase also affects the morphology of mitochondria which are deprived of cristae. We also show that mutants are obligate phototrophs and that rearrangements of the photosynthetic apparatus occur in the chloroplast as a response to ATP synthase deficiency in mitochondria. Altogether, our results contribute to the understanding of the yet poorly studied bioenergetic interactions between organelles in photosynthetic organisms.     

Nucleotides modulate the activity of aspartate racemase of Scapharca broughtonii

The activity of -aspartate racemase purified from Scapharca broughtonii has been found to depend markedly on some nucleotides. Purine nucleoside monophosphates enhanced the enzyme activity, which was, on the contrary, lowered by purine nucleoside triphosphates and not affected by pyrimidine nucleotides. AMP produced the highest increase of seven-fold in the enzyme activity at 6 mM and a half-maximum increase at approximately 3.8 mM. ATP caused a half-maximum decrease in the activity at approximately 1.4 mM and the remaining activity was lower than 7% at saturating ATP concentrations. AMP and ATP both brought about changes in V_max and not in K_m. Analysis of the effect of AMP and ATP suggests that each of them has its own primary binding site, which is different from the substrate-binding site. In view of these effects of the nucleotides, the roles of the racemase and -aspartate in energy metabolism under anoxic conditions are discussed.     

ATP synthase in mycobacteria: Special features and implications for a function as drug target

ATP synthase is a ubiquitous enzyme that is largely conserved across the kingdoms of life. This conservation is in accordance with its central role in chemiosmotic energy conversion, a pathway utilized by far by most living cells. On the other hand, in particular pathogenic bacteria whilst employing ATP synthase have to deal with energetically unfavorable conditions such as low oxygen tensions in the human host, e.g. Mycobacterium tuberculosis can survive in human macrophages for an extended time. It is well conceivable that such ATP synthases may carry idiosyncratic features that contribute to efficient ATP production. In this review genetic and biochemical data on mycobacterial ATP synthase are discussed in terms of rotary catalysis, stator composition, and regulation of activity. ATP synthase in mycobacteria is of particular interest as this enzyme has been validated as a target for promising new antibacterial drugs. A deeper understanding of the working of mycobacterial ATP synthase and its atypical features can provide insight in adaptations of bacterial energy metabolism. Moreover, pinpointing and understanding critical differences as compared with human ATP synthase may provide input for the design and development of selective ATP synthase inhibitors as antibacterials. This article is part of a Special Issue entitled: 18th European Bioenergetic Conference.     

Cloning and biochemical characterization of a novel mouse ADP-dependent glucokinase

Glycolysis, the catabolism of glucose to pyruvate, is an iconic central metabolic pathway and often used as a paradigm for explaining the general principles of the regulation/control of cellular metabolism. The ubiquitous mammalian ATP-dependent hexokinases I-III and hexokinase IV, also termed glucokinase, initiate the process by phosphorylating glucose to glucose-6-phosphate. Despite glycolysis having been studied extensively for over 70 years and the last new mammalian ATP-dependent hexokinase isotype having been described in the 1960s, we report here the biochemical characterization of a recombinant ADP-dependent glucokinase cloned from a full-length Mus musculus cDNA, identified by sequence analysis. The recombinant enzyme is quite specific for glucose, is monomeric, has an apparent Km for glucose and ADP of 96 and 280 microM, respectively, and is inhibited by both high concentrations of glucose and AMP. The metabolic role of this enzyme in cells would be dependent on the relative level of its activity to those of the ATP-dependent hexokinases. The greatest advantage of an ADP-GK would clearly be during ischemia/hypoxia, clinically relevant conditions in multiple major disease states, by decreasing the priming cost for the phosphorylation of glucose, saving ATP.     

The mechanism of rotating proton pumping ATPases

Two proton pumps, the F-ATPase (ATP synthase, F_oF₁) and the V-ATPase (endomembrane proton pump), have different physiological functions, but are similar in subunit structure and mechanism. They are composed of a membrane extrinsic (F₁ or V₁) and a membrane intrinsic (F_o or V_o) sector, and couple catalysis of ATP synthesis or hydrolysis to proton transport by a rotational mechanism. The mechanism of rotation has been extensively studied by kinetic, thermodynamic and physiological approaches. Techniques for observing subunit rotation have been developed. Observations of micron-length actin filaments, or polystyrene or gold beads attached to rotor subunits have been highly informative of the rotational behavior of ATP hydrolysis-driven rotation. Single molecule FRET experiments between fluorescent probes attached to rotor and stator subunits have been used effectively in monitoring proton motive force-driven rotation in the ATP synthesis reaction. By using small gold beads with diameters of 40-60 nm, the E. coli F₁ sector was found to rotate at surprisingly high speeds (> 400 rps). This experimental system was used to assess the kinetics and thermodynamics of mutant enzymes. The results revealed that the enzymatic reaction steps and the timing of the domain interactions among the β subunits, or between the β and γ subunits, are coordinated in a manner that lowers the activation energy for all steps and avoids deep energy wells through the rotationally-coupled steady-state reaction. In this review, we focus on the mechanism of steady-state F₁-ATPase rotation, which maximizes the coupling efficiency between catalysis and rotation.     

Na+ Transport by the A1AO-ATP Synthase Purified from Thermococcus onnurineus and Reconstituted into Liposomes

The ATP synthase of many archaea has the conserved sodium ion binding motif in its rotor subunit, implying that these A₁A_O-ATP synthases use Na⁺ as coupling ion. However, this has never been experimentally verified with a purified system. To experimentally address the nature of the coupling ion, we have purified the A₁A_O-ATP synthase from T. onnurineus. It contains nine subunits that are functionally coupled. The enzyme hydrolyzed ATP, CTP, GTP, UTP, and ITP with nearly identical activities of around 40 units/mg of protein and was active over a wide pH range with maximal activity at pH 7. Noteworthy was the temperature profile. ATP hydrolysis was maximal at 80 °C and still retained an activity of 2.5 units/mg of protein at 45 °C. The high activity of the enzyme at 45 °C opened, for the first time, a way to directly measure ion transport in an A₁A_O-ATP synthase. Therefore, the enzyme was reconstituted into liposomes generated from Escherichia coli lipids. These proteoliposomes were still active at 45 °C and coupled ATP hydrolysis to primary and electrogenic Na⁺ transport. This is the first proof of Na⁺ transport by an A₁A_O-ATP synthase and these findings are discussed in light of the distribution of the sodium ion binding motif in archaea and the role of Na⁺ in the bioenergetics of archaea.     

A role for anions in ATP synthesis and its molecular mechanistic interpretation

ATP, the ‘universal biological energy currency’, is synthesized by utilizing energy either from oxidation of fuels or from light, via the process of oxidative and photo-phosphorylation respectively. The process is mediated by the enzyme F₁F₀-ATP synthase, using the free energy of ion gradients in the final energy catalyzing step, i.e., the synthesis of ATP from ADP and inorganic phosphate (P_i). The details of the molecular mechanism of ATP synthesis are among the most important fundamental issues in biology and hence need to be properly understood. In this work, a role for anions in making ATP has been found. New experimental data has been reported on the inhibition of ATP synthesis at nanomolar concentrations by the potent, specific anion channel blockers 4,4′-diisothiocyanostilbene-2, 2′-disulphonic acid (DIDS) and tributyltin chloride (TBTCl). Based on these inhibition studies, attention has been drawn to anion translocation (in addition to proton translocation) as a requirement for ATP synthesis. The type of inhibition has been quantified and an overall kinetic scheme for mixed inhibition that explains the data has been evolved. The experimental data and the type of inhibition found have been interpreted in the light of the torsional mechanism of energy transduction and ATP synthesis (Nath J Bioenerg Biomembr 42:293–300, 2010a; J Bioenerg Biomembr 42:301–309, 2010b). This detailed and unified mechanism resolves long-standing problems and inconsistencies in the first theories (Slater Nature 172:975–978, 1953; Williams J Theor Biol 1:1–17, 1961; Mitchell Nature 191:144–148, 1961; Mitchell Biol Rev 41:445–502, 1966), makes several novel predictions that are experimentally verifiable (Nath Biophys J 90:8–21, 2006a; Process Biochem 41:2218–2235, 2006b), and provides us with a new and fruitful paradigm in bioenergetics. The interpretation presented here provides intelligent answers to the unexplained existing results in the literature. It is shown that mechanistic interpretation of the experimental data requires substantial addition to available conceptual foundations such that present concepts, theories, and mechanisms must be revised.     

A 31P-NMR saturation transfer study of the regulation of creatine kinase in the rat heart

(1) ³¹P nuclear magnetic resonance was used to measure the creatine kinase-catalysed fluxes in Langendorff-perfused rat hearts consuming oxygen at different rates and using either of two exogenous substrates (11 mM glucose or 5 mM acetate). (2) Fluxes in the direction of ATP synthesis were between 3.5–12-times the steady-state rates of ATP utilization (estimated from rates of O₂-consumption), demonstrating that the reaction is sufficiently rapid to maintain the cytosolic reactants near their equilibrium concentrations. (3) Under all conditions studied, the cytosolic free [ADP] was primarily responsible for regulating the creatine kinase fluxes. The enzyme displayed a K_m for cytosolic ADP of 35 μM and an apparent V_max of 5.5 mM/s in the intact tissue. (4) Although the reaction is maintained in an overall steady-state, the measured ratio of the forward flux (ATP synthesis) to the reverse flux (phosphocreatine synthesis) was significantly greater than unity under some conditions. It is proposed that this discrepancy may be a consequence of participation of ATP in reactions other than the PCr /ag ATP or ATP /ag ADP + P_i interconversions specifically considered in the analysis. (5) The results support the view that creatine kinase functions primarily to maintain low cytosolic concentrations of ADP during transient periods in which energy utilization exceeds production.     

Altered expression of the chloroplast ATP synthase through site-directed mutagenesis in <Emphasis Type="Italic">Chlamydomonas reinhardtii</Emphasis>

The chloroplast ATP synthase gates the flow of protons out of the thylakoid lumen. In Chlamydomonas reinhardtii deletion of any of the genes for the ATP synthase subunits, or misfolding of the peptides results in photosynthetic membranes devoid of the enzyme (Lemaire and Wollman, J Biol Chem 264:675–685, 1989). This work examines the physiologic response of an algal strain in which the epsilon subunit of the chloroplast ATP synthase has been truncated. Removal of 10 amino acids from the C-terminus of the peptide results in a sharp decrease in the content of the enzyme, but does not result in its exclusion from the thylakoid membranes. The ATP synthase of this mutant strain has a higher rate of ATP hydrolysis than the wild-type enzyme. This strain of C. reinhardtii exhibits reduced growth in the light, dependence on acetate, and a low threshold for the onset of photoinhibition. The role of the ATP synthase in regulating the proton concentration of the lumen is discussed. This work was supported in part by a grant from the National Science Foundation (MCB0110232).     

The effect of hormones on metal and metal-ATP interactions with fat cell adenylate cyclase.

1-adrenaline, ACTH and glucagon activate the adenylate cyclase of rat adipocytes by decreasing its S_0.5(Mg²⁺) (concentration yielding 0.5 V_max) from its basal value of 11.5 to 1.2, 0.3 and 1.8 mM and by increasing its K_i(ATP^4?) from 0.03 to 0.25; 0.62 and 0.16 mM respectively. The kinetic properties of the enzyme are regulated by its state of saturation with ATP^4? or Mg²⁺; its saturation with ATP^4? and citrate^3? suppressed its basal and hormone-dependent activities. The hormone-dependent decrease in K_m and increase in V_max of the enzyme occur when shifting from suboptimal low concentrations of hormone and Mg²⁺ to optimal conditions, i.e., high concentration of hormone and low concentration of Mg²⁺. The increase in the state of saturation of the enzyme with Mg²⁺ decreases the hormone-dependent effects on V_max and results in identical values of K_m (0.14 mM) for its basal and 1-adrenaline dependent activities. CaCl₂ saturation curves at 5 mM ATP with either 5, 10 or 20 mM MgCl₂ show that the substitution of 5 mM MgCl₂ by 10 mM and 20 mM MgCl₂ increased the K_i(Ca²⁺) of the enzyme from 0.19 to 0.49 and 0.94 mM but decreased its K_i(CaATP) from 0.42 to 0.19 and 0.14 mM respectively. Only when the concentration of MgCl₂ exceeded that of ATP did 1-adrenaline and ACTH activate the enzyme by increasing its K_i(Ca²⁺), although only ACTH increased its K_i(CaATP). An increase in energy charge would decrease the intracellular concentrations of Mg²⁺ and Ca²⁺ because ATP^4? has stronger binding constants for Mg²⁺ and Ca²⁺ than ADP^3? and AMP^2?. Hence, the reported properties of the enzyme suggests that changes in energy charge may allow for metabolic feedback control of the hormonal responsiveness of the Mg²⁺, Ca²⁺, ATP^4? -sensitive adenylate cyclase.     

Characterization of ATP release from cultures enriched in cholinergic amacrine‐like neurons

Adenosine triphosphate (ATP) has been proposed to play a role as a neurotransmitter in the retina, but not much attention has been given to the regulation of ATP release from retinal neurons. In this work, we investigated the release of ATP from cultures enriched in amacrine‐like neurons. Depolarization of the cells with KCl, or activation of α‐amino‐3‐hydroxy‐ 5‐methyl‐4‐isoxazole‐propionate (AMPA) receptors, evoked the release of ATP, as determined by the luciferin/luciferase luminescent method. The ATP release was found to be largely Ca²⁺ dependent and sensitive to the botulinum neurotoxin A, which indicates that the ATP released by cultured retinal neurons originated from an exocytotic pool. Nitrendipine and ω‐Agatoxin IVA, but not by ω‐Conotoxin GVIA, partially blocked the release of ATP, indicating that in these cells, the Ca²⁺ influx necessary to trigger the release of ATP occurs in part through the L‐ and the P/Q types of voltage‐sensitive Ca²⁺ channels (VSCC), but not through N‐type VSCC. The release of ATP increased in the presence of adenosine deaminase, or in the presence of 1,3‐dipropyl‐8‐cyclopentylxanthine (DPCPX), an adenosine A₁ receptor antagonist, showing that the release is tonically inhibited by the adenosine A₁ receptors. To our knowledge, this is the first report showing the release of endogenous ATP from a retinal preparation. © 1999 John Wiley & Sons, Inc. J Neurobiol 41: 340–348, 1999     

Mitochondrial calcium as a key regulator of mitochondrial ATP production in mammalian cells

Mitochondrial Ca²⁺ transport was initially considered important only in buffering of cytosolic Ca²⁺ by acting as a “sink” under conditions of Ca²⁺ overload. The main regulator of ATP production was considered to be the relative concentrations of high energy phosphates. However, work by Denton and McCormack in the 1970s and 1980s showed that free intramitochondrial Ca²⁺ ([Ca²⁺]_m) activated dehydrogenase enzymes in mitochondria, leading to increased NADH and hence ATP production. This leads them to propose a scheme, subsequently termed a “parallel activation model” whereby increases in energy demand, such as hormonal stimulation or increased workload in muscle, produced an increase in cytosolic [Ca²⁺] that was relayed by the mitochondrial Ca²⁺ transporters into the matrix to give an increase in [Ca²⁺]_m. This then stimulated energy production to meet the increased energy demand. With the development of methods for measuring [Ca²⁺]_m in living cells that proved [Ca²⁺]_m changed over a dynamic physiological range rather than simply soaking up excess cytosolic [Ca²⁺], this model has now gained widespread acceptance. However, work by ourselves and others using targeted probes to measure changes in both [Ca²⁺] and [ATP] in different cell compartments has revealed variations in the interrelationships between these two in different tissues, suggesting that metabolic regulation by Ca²⁺ is finely tuned to the demands and function of the individual organ.     

Supramolecular organization of the yeast F1Fo-ATP synthase

Background information. The yeast mitochondrial F₁F_o‐ATP synthase is a large complex of 600 kDa that uses the proton electrochemical gradient generated by the respiratory chain to catalyse ATP synthesis from ADP and P_i. For a large range of organisms, it has been shown that mitochondrial ATP synthase adopts oligomeric structures. Moreover, several studies have suggested that a link exists between ATP synthase and mitochondrial morphology. Results and discussion. In order to understand the link between ATP synthase oligomerization and mitochondrial morphology, more information is needed on the supramolecular organization of this enzyme within the inner mitochondrial membrane. We have conducted an electron microscopy study on wild‐type yeast mitochondria at different levels of organization from spheroplast to isolated ATP synthase complex. Using electron tomography, freeze‐fracture, negative staining and image processing, we show that cristae form a network of lamellae, on which ATP synthase dimers assemble in linear and regular arrays of oligomers. Conclusions. Our results shed new light on the supramolecular organization of the F₁F_o‐ATP synthase and its potential role in mitochondrial morphology.     

Enzymatic studies on the interaction of myosin and heavy meromyosin with 1,N 6 -ethenoadenosine triphosphate ( ATP), a fluorescent analog of ATP

The fluorescent analog of adenosine triphosphate (ATP)¹ 1,N⁶-ethenoadenosine triphosphate, (εATP), has been utilized as a substitute for ATP in the myosin and heavy meromyosin ATPase systems. For myosin, the analog εATP replaced ATP with a somewhat larger K_m (2.6 × 10^?4 mole ?^?1 for εATP as opposed to 8.8 × 10^?5 mole ?^?1 for ATP), indicating that the apparent affinity of the enzyme for εATP is less than for ATP. Perhaps of more interest, further comparison yielded a V_max for εATP about two and one half times the value for ATP (20 μmole PO₄ sec^?1 g protein^?1 as opposed to 8.1 μmole sec^?1 g protein^?1). Results for the HMM-εATPase system were similar, yielding a K_m value of 1.47 × 10^?4 mole ?^?1 and a V_max of 54.2 μmole PO₄ sec^?1 g protein^?1, as opposed to corresponding K_m and V_max values of 1.23 × 10^?4 mole ?^?1 and 20.4 μmole PO₄ sec^?1 g protein^?1, respectively for the HMM-ATP interaction. The pH dependence of εATPase for both systems was comparable to ATP, suggesting a similarity in the mechanism of hydrolysis of the two nucleotides. Activation of εATPase by Ca²⁺ in the presence of 0.5 M KCl was comparable to ATPase for both systems, but inhibition by Mg²⁺ seemed to be more effective for εATPase. These results indicate that εATP is an excellent substitute for ATP in the myosin and heavy meromyosin systems and because of its insertion into the active site of these muscle proteins, it promises to be a very useful probe for conformation studies at this level.     

Purification and characterization of an ADP-dependent phosphofructokinase from Thermococcus zilligii

The ADP-dependent phosphofructokinase (PFK) from Thermococcus zilligii has been purified 950 fold; it had a specific activity of 190 U mg⁻¹. The enzyme required Mg²⁺ ions for optimal activity and was specific for ADP. The forward reaction kinetics were hyperbolic for both cosubstrates (pH optimum of 6.4), and the apparent K _m values for ADP and fructose-6-phosphate were 0.6 mM (apparent V _max of 243 U mg⁻¹) and 1.47 mM (apparent V _max of 197 U mg⁻¹), respectively. Significantly, the enzyme is indicated to be nonallosteric but was slightly activated by some monovalent cations including Na⁺ and K⁺. The protein had a subunit size of 42.2 kDa and an estimated native molecular weight of 66 kDa (gel filtration). Maximal reaction rates for the reverse reaction were attained at pH 7.5–8.0, and the apparent K _m values for fructose-1,6-bisphosphate and AMP were 0.56 mM (apparent V _max of 2.9 U mg⁻¹) and 12.5 mM, respectively. The biochemical characteristics of this unique ADP-dependent enzymatic activity are compared to ATP and pyrophosphate-dependent phosphofructokinases. Received: August 14, 1998 / Accepted: December 2, 1998     

Characterization of a highly diverged mitochondrial ATP synthase Fo subunit in Trypanosoma brucei

The mitochondrial F₁F_o ATP synthase of the parasite Trypanosoma brucei has been previously studied in detail. This unusual enzyme switches direction in functionality during the life cycle of the parasite, acting as an ATP synthase in the insect stages, and as an ATPase to generate mitochondrial membrane potential in the mammalian bloodstream stages. Whereas the trypanosome F₁ moiety is relatively highly conserved in structure and composition, the F_o subcomplex and the peripheral stalk have been shown to be more variable. Interestingly, a core subunit of the latter, the normally conserved subunit b, has been resistant to identification by sequence alignment or biochemical methods. Here, we identified a 17 kDa mitochondrial protein of the inner membrane, Tb927.8.3070, that is essential for normal growth, efficient oxidative phosphorylation, and membrane potential maintenance. Pull-down experiments and native PAGE analysis indicated that the protein is both associated with the F₁F_o ATP synthase and integral to its assembly. In addition, its knockdown reduced the levels of F_o subunits, but not those of F₁, and disturbed the cell cycle. Finally, analysis of structural homology using the HHpred algorithm showed that this protein has structural similarities to F_o subunit b of other species, indicating that this subunit may be a highly diverged form of the elusive subunit b.