Selective labelling of phosphorylase kinase with fluorescein isothiocyanate

Fluorescein isothiocyanate (FITC) is a highly specific inhibitor of rabbit muscle phosphorylase kinase. The rapid inhibition process is accompanied by an almost exclusive incorporation of fluorescein into the α sub-unit. A molar ratio of 0.8 mol FITC per mol α subunit for a 60% inhibited kinase was calculated. Mg²⁺ and Mg²⁺-ATP completely block the inhibitory effect of FITC, but ATP, ADP and Ca²⁺ have no significant effect on FITC inhibition. Trypsin-activated phosphorylase kinase is not inactivated by FITC, while the fluorescein-modified enzyme can be activated by digestion with trypsin to the same level of activity of trypsin-activated unmodified enzyme.     

Interactions involved in grasping and locking of the inhibitory peptide IF1 by mitochondrial ATP synthase

When mitochondria become deenergized, futile ATP hydrolysis is prevented by reversible binding of an endogenous inhibitory peptide called IF1 to ATP synthase. Between initial IF1 binding and IF1 locking the enzyme experiences large conformational changes. While structural studies give access to analysis of the dead-end inhibited state, transient states have thus far not been described. Here, we studied both initial and final states by reporting, for the first time, the consequences of mutations of Saccharomyces cerevisiae ATP synthase on its inhibition by IF1. Kinetic studies allowed the identification of amino acids or motifs of the enzyme that are involved in recognition and/or locking of IF1 α-helical midpart. This led to an outline of IF1 binding process. In the recognition step, protruding parts of α and especially β subunits grasp IF1, most likely by a few residues of its α-helical midpart. Locking IF1 within the αβ interface involves additional residues of both subunits. Interactions of the α and β subunits with the foot of the γ subunit might contribute to locking and stabilizing of the dead-end state.     

The use of 8-azido-ATP and 8-azido-ADP as photoaffinity labels of the ATP synthase in submitochondrial particles: Evidence for a mechanism of ATP hydrolysis involving two independent catalytic sites?

8-Azido-ATP is a substrate for the ATP synthase in submitochondrial particles with a V_max equal to 6% of the V_max with ATP. The K_m values for 8-azido-ATP are similar to those for ATP. ATP synthase in submitochondrial particles can bind maximally 2 mol 8-N-ATP or 8-N-ADP per mole and the inhibition of ATP hydrolysis by covalently bound N-ATP or N-ADP is proportional to the saturation of the enzyme with inhibitor, similar to the results obtained with isolated F₁. Both 8-N-ATP and 8-N-ADP are bound mainly to the β subunits and at all levels of saturation the distribution of the label is 77% to the β and 23% to the α subunits. It is proposed that the binding of 8-azido-AXP itself is mainly to the β subunit, but that part of the nitreno radicals formed during excitation with light reacts with an amino acid of the α subunit, due to the location of the binding site at an interface between a β and an α subunit. Partial saturation with 8-N-ATP, under conditions that the concentration of 8-azido-ATP during the incubation is intermediate between the low and high K_m values, does not abolish the apparent negative cooperativity of ATP hydrolysis. It is concluded that this apparent cooperativity is not due to the presence of two different catalytic sites, nor to a cooperativity between the two catalytic sites, but to interaction between the catalytic sites and regulatory sites.     

Neuronal cell surface ATP synthase mediates synthesis of extracellular ATP and regulation of intracellular pH

The ATP synthase is known to play important roles in ATP generation and proton translocation within mitochondria. Here, we now provide evidence showing the presence of functional ecto‐ATP synthase on the neuronal surface. Immunoblotting revealed that the α, β subunits of ATP synthase F₁ portion are present in isolated fractions of plasma membrane and biotin‐labelled surface protein from primary cultured neurons; the surface distribution of α, β subunits was also confirmed by immunofluorescence staining. Moreover, α and β subunits were also found in fractions of plasma membrane and lipid rafts isolated from rat brain, and flow cytometry analysis showed α subunits on the surface of acutely isolated brain cells. Activity assays showed that the extracellular ATP generation of cultured neurons could be compromised by α, β subunit antibodies and ATP synthase inhibitors. pH_i (intracellular pH) analysis demonstrated that at low extracellular pH, α or β subunit antibodies decreased pH_i of primary cultured neurons. Therefore, ATP synthase on the surface of neurons may be involved in the machineries of extracellular ATP generation and pH_i homoeostasis.     

Knockdown of F1 epsilon subunit decreases mitochondrial content of ATP synthase and leads to accumulation of subunit c

The subunit ε of mitochondrial ATP synthase is the only F₁ subunit without a homolog in bacteria and chloroplasts and represents the least characterized F₁ subunit of the mammalian enzyme. Silencing of the ATP5E gene in HEK293 cells resulted in downregulation of the activity and content of the mitochondrial ATP synthase complex and of ADP-stimulated respiration to approximately 40% of the control. The decreased content of the ε subunit was paralleled by a decrease in the F₁ subunits α and β and in the F_o subunits a and d while the content of the subunit c was not affected. The subunit c was present in the full-size ATP synthase complex and in subcomplexes of 200–400 kDa that neither contained the F₁ subunits, nor the F_o subunits. The results indicate that the ε subunit is essential for the assembly of F₁ and plays an important role in the incorporation of the hydrophobic subunit c into the F₁-c oligomer rotor of the mitochondrial ATP synthase complex.     

Indications for an oligomeric structure and for conformational changes in sarcoplasmic reticulum Ca2+-ATPase labelled selectively with fluorescein

Fluorescein isothiocyanate is a highly specific inhibitor of the Ca2+-ATPase from sarcoplasmic reticulum. The Ca2+ pumping is inhibited completely at a fluorescein isothiocyanate concentration half that of the ATPase protein, indicating that the protein is at least a dimer. ATP protected specifically against fluorescein isothiocyanate inhibition, indicating that fluorescein isothiocyanate may react at the nucleotide binding site of the ATPase (probably with a reactive lysine residue). The fluorescein is incorporated almost exclusively into the 105 kdalton catalytic polypeptide of the ATPase and digestion by trypsin gives rise to a fluorescein-labelled 45 kdalton fragment. Conformational changes induced by addition of Ca can be studied conveniently with the fluorescein-labelled ATPase.     

Subunit-subunit interactions in TF1 as revealed by ligand binding to isolated and integrated α and β subunits

The binding of 3′-O-(1-naphthoyl)adenosinetriphosphate (1-naphthoyl-ATP), ATP and ADP to TF₁ and to the isolated α and β subunits was investigated by measuring changes of intrinsic protein fluorescence and of fluorescence anisotropy of 1-naphthoyl-ATP upon binding. The following results were obtained. (1) The isolated α and β subunits bind 1 mol 1-naphthoyl-ATP with a dissociation constant (K_D(1-naphthoyl-ATP)) of 4.6 μM and 1.9 μM, respectively. (2) The K_D(ATP) for α and β subunits is 8 μM and 11 μM, respectively. (3) The K_D(ADP) for α and β subunits is 38 μM μM and 7 μM, respectively. (4) TF₁ binds 2 mol 1-naphthoyl-ATP per mol enzyme with K_D = 170 nM. (5) The rate constant for 1-naphthoyl-ATP binding to α and β subunit is more than 5 · 10⁴ M⁻¹s⁻¹. (6) The rate constant for 1-naphthoyl-ATP binding to TF₁ is 6.6 · 10³ M⁻¹ · s⁻¹ (monophasic reaction); the rate constant for its dissociation in the presence of ATP is biphasic with a fast first phase (k^A₋₁ = 3 · 10⁻³s⁻¹) and a slower second phase (k^A₋₂ < 0.2 · 10⁻³s⁻¹). From the appearance of a second peak in the fluorescence emission spectrum of 1-naphthoyl-ATP upon binding it is concluded that the binding sites in TF₁ are located in an environment more hydrophobic than the binding sites on isolated α and β subunits. The differences in kinetic and thermodynamic parameters for ligand binding to isolated versus integrated α and β subunits, respectively, are explained by interactions between these subunits in the enzyme complex.     

Reconstitution of ATPase activity from the isolated alpha, beta, and gamma subunits of the coupling factor, F1, of Escherichia coli.

The three major subunits (α, β and γ) of the coupling factor, F₁ ATPase, of $\text{Escherichia coli}$ were separated and purified by hydrophobic column chromatography after the enzyme was dissociated by cold inactivation. The ability to hydrolyze ATP was reconstituted by dialyzing the mixture of subunits against 0.05 M Tris-succinate, pH 6.0, containing 2 mM ATP and 2 mM MgCl₂. A mixture containing α, β and γ regained ATP hydrolyzing activity. Individual subunits alone or mixtures of any two subunits did not develop ATPase activity, except for a low but significant activity with α plus β. The reconstituted ATPase had a K_m of 0.23 mM for ATP and a molecular weight by sucrose gradient density centrifugation of about 280,000.     

Inhibition and labeling of sodium, potassium ATPase by the dialdehyde derivative of ATP

Canine renal Na,K-ATPase was treated with ATP dialdehyde, "oxATP" (20 microM), as described by G. Ponzio, B. Rossi, and M. Lazdunski (1983, J. Biol. Chem. 258, 8201-8205). In this system, a by-product, formaldehyde, was the inactivator. We modified the system to minimize such inhibition and to speed up the reaction. oxATP itself inactivated the enzyme at a rate that was slow at first and later speeded up. We fitted a precursor-product model to the data. Labeling with [3H]oxATP indicated about three sites per alpha beta protomer at complete inactivation. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the labeled enzyme showed radioactivity in many components, in the alpha and beta subunits and in small molecules at the tracker dye region. ATP (20 mM) prevented all labeling and inactivation. Ponzio et al. concluded that oxATP labels covalently an ATP binding site. Our experiments did not support this conclusion. Ouabain did not affect labeling. Sodium stimulated both inhibition and labeling more than potassium did, indicating a high-affinity ATP binding site, if any. But nucleotide specificity for preventing or producing inhibition did not correspond to nucleotide specificity for binding of ATP to the native enzyme. Blocking the ATP binding center with fluorescein isothiocyanate or fluorosulfonyl benzoyl adenosine had no effect on [3H]oxATP labeling. ATP also prevented [3H]oxATP labeling of bovine serum albumin or of integral-membrane proteins.     

Nucleotide specificity of cardiac sarcoplasmic reticulum. Inhibition of GTPase activity by ATP analogue in fluorescein isothiocyanate-modified calcium ATPase.

Unlike skeletal muscle sarcoplasmic reticulum, canine cardiac sarcoplasmic reticulum hydrolyzes GTP in ways that are similar and different from ATP hydrolysis. Also, ATP and ATP analogues inhibit GTPase activity noncompetitively with a Ki compatible with the high affinity ATP-binding site (c.f. Tate, C.A., Bick, R.J., Blaylock, S., Youker, K., Scherer, N.M., and Entman, M.L. (1989) J. Biol. Chem. 264, 7809-7813). This suggested that ATP and GTP may enter the reaction pathway at separate nucleotide-binding sites on the CaATPase. To test this hypothesis, cardiac sarcoplasmic reticulum was incorporated with fluorescein isothiocyanate (FITC), which apparently binds at or near the ATP-binding site of the enzyme, preventing ATP binding. After FITC incorporation, calcium-dependent ATPase activity, but not GTPase activity, was completely inhibited. Adenyl-5'-yl imidodiphosphate (AMP-P(NH)P), but not guanyl-5'-yl imidodiphosphate, protected against FITC incorporation and the inhibition of calcium-dependent ATPase activity; at least 100 microM AMP-P(NH)P was required for some protection. Despite FITC incorporation, AMP-P(NH)P still inhibited the GTPase activity with a Ki of 3-7 microM. Direct photo-affinity labeling with either 0.2 microM [alpha-32P]ATP or 0.2 microM [alpha-32P]GTP demonstrated that FITC incorporation did not prevent ATP or GTP binding. The mechanism of FITC inhibition of calcium-dependent ATPase activity was related to the prevention of all calcium-dependent, but not calcium-independent, reactions with both nucleotides.     

Mechanism of allosteric regulation of the Ca,Mg-ATPase of sarcoplasmic reticulum: studies with 5'-adenylyl methylenediphosphate

Four mechanisms for the allosteric regulation of the calcium and magnesium ion activated adenosinetriphosphatase (Ca,Mg-ATPase) of sarcoplasmic reticulum were examined. Negative cooperativity in substrate binding was not supported by 3H-labeled 5'-adenylyl methylenediphosphate (AMPPCP) binding, which was best fit by a single class of sites. Although calcium had no effect on the absence of cooperativity, it did increase the affinity of the enzyme for AMPPCP. Allosteric regulation via an effector site for AMPPCP or ATP on the same ATPase chain was eliminated by the stoichiometry of ATP and AMPPCP binding, 1 mol of site per mole of enzyme. The possibility that AMPPCP acts at an effector site was eliminated by showing that it competitively inhibits the rate of phosphoenzyme formation. Allosteric regulation of kinetics via site-site interaction in an oligomer was eliminated by showing that the inhibition of ATPase activity by fluorescein isothiocyanate is linearly dependent upon its incorporation into the sarcoplasmic reticulum. The fourth mechanism considered was stimulation of ATPase activity by the binding of ATP or AMPPCP at the active site after departure of ADP but before the departure of inorganic phosphate. This hypothesis was supported by site stoichiometry and by the observation that AMPPCP or ATP stimulates v/EP, the rate of ATP hydrolysis for a given level of phosphoenzyme. Computer simulation of this branched monomeric model could duplicate all experimental observations made with AMPPCP and ATP as allosteric regulators. The condition that the affinity of ATP binding to the enzyme be reduced when it is phosphorylated, which is required by the computer model, was confirmed experimentally.     

Adenine nucleotide binding sites in normal and mutant adenosine triphosphatases of Escherichia coli

Three types of assays were used to characterize adenine nucleotide binding sites on the Ca²⁺, Mg²⁺-activated ATPase of normal Escherichia coli and its unc A 401 and unc D 412 mutants. ADP was bound mainly at a single site in normal and mutant ATPase. In the absence of divalent cations ATP was bound at a single high-affinity and three low-affinity sites in normal and unc D ATPases. The 2′,3′-dialdehyde (oADP) obtained by periodate oxidation of ADP reacted with both low- and high-affinity sites whereas oATP was bound primarily at a low-affinity site. Two types of adenine nucleotide binding sites, a high-affinity site reacting with ATP and ADP and a low-affinity site for ATP, were detected by the effects of these nucleotides on the fluorescence of the aurovertin D-ATPase complex. This high-affinity site(s) was present in normal and mutant ATPases. However, the fluorescence response at both high- and low-affinity sites was modified in the unc D ATPase as a consequence of the abnormal β subunit in this enzyme. Normal fluorescence responses were not induced by the binding of oADP or oATP to the ATPases. ATP was bound at a single site on isolated α subunits of the enzyme. Since this site was not detected in the unc A ATPase, it is unlikely to be the high-affinity site detected in the intact enzyme or the binding site for the endogenous tightly bound adenine nucleotides found in the purified ATPase. It is more probable that the site detected on the isolated α subunit from the normal enzyme is that which binds oADP since this site was absent in the unc A ATPase. Pretreatment of the normal ATPase with either N, N′-dicyclohexyl-carbodiimide (DCCD) or with 4-chloro-7-nitrobenzofurazan (NbfCl), reagents which inhibit ATPase activity by reacting with a β subunit, affected binding of oADP to α subunit(s) but had less effect with oATP. Inhibition of oADP binding could be due to conformational changes induced in the α subunit by the reaction of DCCD and NbfCl with a β subunit, or to steric reasons. If the latter hypothesis is correct, the active site of the ATPase would be at the interface between α and β subunits of the enzyme.     

Allosteric property of the (Na⁺+K⁺)-ATPase β₁ subunit

(Na(+)+K(+))-ATPase (NKA) comprises two basic α and β subunits: The larger α subunit catalyzes the hydrolysis of ATP for active transport of Na(+) and K(+) ions across the plasma membrane; the smaller β subunit does not take part in the catalytic process of the enzyme. Little is known about allosteric regulation of the NKA β subunit. Here, we report a surprising finding that extracellular stimuli on the native β(1) subunit can generate a significant impact on the catalytic function of NKA. By using a β(1) subunit-specific monoclonal antibody JY2948, we found that the JY2948-β(1) subunit interaction markedly enhances the catalytic activity of the enzyme and increases the apparent affinity of Na(+) and K(+) ions for both ouabain-resistant rat NKA and ouabain-sensitive dog NKA. This study provides the first evidence to identify an allosteric binding site residing on the NKA β(1) subunit and uncovers the latent allosteric property of the β(1) subunit, which remotely controls the NKA catalytic function.     

Principal role of the arginine finger in rotary catalysis of F1-ATPase

F(1)-ATPase (F(1)) is an ATP-driven rotary motor wherein the γ subunit rotates against the surrounding α(3)β(3) stator ring. The 3 catalytic sites of F(1) reside on the interface of the α and β subunits of the α(3)β(3) ring. While the catalytic residues predominantly reside on the β subunit, the α subunit has 1 catalytically critical arginine, termed the arginine finger, with stereogeometric similarities with the arginine finger of G-protein-activating proteins. However, the principal role of the arginine finger of F(1) remains controversial. We studied the role of the arginine finger by analyzing the rotation of a mutant F(1) with a lysine substitution of the arginine finger. The mutant showed a 350-fold longer catalytic pause than the wild-type; this pause was further lengthened by the slowly hydrolyzed ATP analog ATPγS. On the other hand, the mutant F(1) showed highly unidirectional rotation with a coupling ratio of 3 ATPs/turn, the same as wild-type, suggesting that cooperative torque generation by the 3 β subunits was not impaired. The hybrid F(1) carrying a single copy of the α mutant revealed that the reaction step slowed by the mutation occurs at +200° from the binding angle of the mutant subunit. Thus, the principal role of the arginine finger is not to mediate cooperativity among the catalytic sites, but to enhance the rate of the ATP cleavage by stabilizing the transition state of ATP hydrolysis. Lysine substitution also caused frequent pauses because of severe ADP inhibition, and a slight decrease in ATP-binding rate.     

Effect of dicyclohexylcarbodiimide on unisite and multisite catalytic activities of the adenosinetriphosphatase of Escherichia coli

The inhibitory effect of dicyclohexylcarbodiimide (DCCD) on the activity of the adenosine-triphosphatase of Escherichia coli (ECF1) has been examined in detail. DCCD reacted with ECF1 predominantly in beta subunits with a maximum of 2 mol of reagent per mole of ECF1 being incorporated in these subunits. Ninety-five percent inhibition of steady-state or multistate ATPase activity required incorporation of 1 mol of DCCD per mole of enzyme into beta subunits. Seventy-five percent inhibition of the initial rate of unisite catalysis was only obtained after incorporation of 2 mol of DCCD per mole of ECF1 into beta subunits. Analyses of the kinetics of unisite catalysis and nucleotide binding experiments both indicate that DCCD binds outside the substrate ATP binding site. Inhibition by this reagent appears to be due in part to an effect on the catalytic sites but mainly to the blocking of cooperativity between these sites.     

Nonidentical subunits of pyrocatechase from Pseudomonas arvilla C-1.

Pyrocatechase [catechol:oxygen, 1,2-oxidoreductase (decyclizing), EC 1.13.11.1] from Pseudomonas arvilla C-1 has been reported to contain 2 g atoms of iron/mol of enzyme, based on a molecular weight of 90,000, determined by sedimentation and diffusion constants (Y. Kojima, H. Fujisawa, A. Nakazawa, T. Nakazawa, F. Kanetsuna, H. Taniuchi, M. Nozaki, and O. Hayaishi, 1967, J. Biol. Chem., 242, 3270–3278). The molecular weight was estimated again by sedimentation equilibrium and Sephadex G-200 gel filtration and found to be 63,000 and 60,000, respectively. The enzyme was also found to contain 1 g atom of iron/mol of enzyme, based on a molecular weight of 63,000. The enzyme was dissociated into two bands on polyarcylamide gel electrophoresis in the presence of either sodium dodecyl sulfate or 8 m urea, and was separated into two subunits, α and β, by CM-cellulose chromatography using a buffer solution containing 8 m urea. The molecular weights of the α and β subunits were determined to be 30,000 and 32,000, respectively, by sodium dodecyl sulfate-gel electrophoresis. The NH₂-terminal sequences of these subunits determined by Edman degradation were as follows: α subunit, Thr-Val-Asn-Ile-Ser-His-Thr-Ala-Gln-Ile-Gln-Gln-Phe-Phe-Gln-Gln-(X)-(X)-Gly -Phe-Gly; β subunit, Thr-Val-Lys-Ile-Ser-His-Thr-Ala-Asp-Ile-Gln-Ala-Phe-Phe-Asn-Gln-Val-(X)-Gly-Leu-Asx. The COOH-terminal amino acid residues were determined to be alanine for the α subunit and glycine for the β subunit by three different methods: carboxypeptidase digestion, tritium labeling, and hydrazinolysis. These results indicate that the enzyme consists of two nonidentical subunits, α and β.     

The ATP-binding site of the erythrocyte membrane Ca2+ pump. Amino acid sequence of the fluorescein isothiocyanate-reactive region

The erythrocyte plasma membrane Ca2+-pumping ATPase is known to form an acyl-phosphate catalytic intermediate, but there is otherwise little structural information linking it to the other mammalian ion-pumping ATPases which also form phosphorylated intermediates (the Na+, K+-ATPase of plasma membranes, the Ca2+-ATPase of sarcoplasmic reticulum, and the H+, K+-ATPase of gastric mucosa). We show here that this enzyme possesses a fluorescein isothiocyanate-reactive region similar to that possessed by these other ATPases. Low concentrations (10 microM) of fluorescein isothiocyanate inhibit the ATPase activity of this pump, and this inhibition is prevented by 4 mM ATP. ATP also inhibits the reaction of fluorescein isothiocyanate with a single amino acid residue on the 138-kDa polypeptide chain. A tryptic fragment containing the fluorescein-conjugated residue was isolated by high pressure liquid chromatography. The sequence of this peptide was determined to be NH2-Met1-Tyr2-Ser3-Lys4-Gly5-Ala6-Ser7-Glu8++ +-Ile9-Ile10-Leu11-Arg12-COOH; fluorescein isothiocyanate reacts with the lysine residue. The identities of residues 4-8 are the same as those in a sequence common to the other ATPases mentioned above, except that serine-7 of this sequence is changed to a proline in those ATPases. This substitution, sometimes not considered a homologous one, is not expected to have a major effect on the secondary structure or polarity of this region. Outside of this 5-residue core region of the fluorescein isothiocyanate-reactive site, the homologies among the different ion-pumping ATPases are limited.     

Reconstitution of ATPase from the isolated subunits of coupling factor F1's of Escherichia coli and thermophilic bacterium PS3

ATPase was reconstituted from mixtures of isolated subunits of coupling factor, F₁ ATPase of E. coli (EF₁) and thermophilic bacterium PS3 (TF₁); ability to hydrolyze ATP was attained from the combination of α and β subunits from EF₁ and γ subunit from TF₁, α and β from TF₁ and γ from EF₁, and α and γ from EF₁ and β from TF₁. The β subunit of TF₁ also could complement the EF₁ from an E. coli mutant defective in this subunit. This is the first demonstration of interspecies in vitro recombination of ATPase activity from isolated subunits.     

Normal‐mode‐based modeling of allosteric couplings that underlie cyclic conformational transition in F1 ATPase

F₁ ATPase, a rotary motor comprised of a central stalk ( γ subunit) enclosed by three α and β subunits alternately arranged in a hexamer, features highly cooperative binding and hydrolysis of ATP. Despite steady progress in biophysical, biochemical, and computational studies of this fascinating motor, the structural basis for cooperative ATPase involving its three catalytic sites remains not fully understood. To illuminate this key mechanistic puzzle, we have employed a coarse‐grained elastic network model to probe the allosteric couplings underlying the cyclic conformational transition in F₁ ATPase at a residue level of detail. We will elucidate how ATP binding and product (ADP and phosphate) release at two catalytic sites are coupled with the rotation of γ subunit via various domain motions in α ₃ β ₃ hexamer (including intrasubunit hinge‐bending motions in β subunits and intersubunit rigid‐body rotations between adjacent α and β subunits). To this end, we have used a normal‐mode‐based correlation analysis to quantify the allosteric couplings of these domain motions to local motions at catalytic sites and the rotation of γ subunit. We have then identified key amino acid residues involved in the above couplings, some of which have been validated against past studies of mutated and γ ‐truncated F₁ ATPase. Our finding strongly supports a binding change mechanism where ATP binding to the empty catalytic site triggers a series of intra‐ and intersubunit domain motions leading to ATP hydrolysis and product release at the other two closed catalytic sites. Proteins 2009. © 2009 Wiley‐Liss, Inc.