Energy transduction in photosynthetic bacteria. VIII. Activation of the energy-transducing ATPase by inorganic phosphate.

ATPase activity and ATP-induced energization of photosynthetic membranes from Rhodopseudomonas capsulata are stimulated by phosphate; the maximum stimulatory effect occurs at a concentration between 1 and 2 mM. The sensitivity of the ATPase to oligomycin increases in the presence of phosphate since all the Pi-stimulated activity is inhibited by this antibiotic. Aurovertin, which has no effect on ATPase in the absence of phosphate, inhibits completely the activity elicited by this anion. The addition of Pi induces a substantial increase in the V of ATPase activity without changing the affinity of the enzyme for ATP or ADP. Arsenate, at the same concentrations, produces effects very similar to those of phosphate. The stimulation by arsenate of the transfer of energy from ATP to the membrane suggests a non-hydrolytic role of this anion as a modifier of the ATPase activity.     

Mechanism of Mss116 ATPase reveals functional diversity of DEAD-Box proteins

Mss116 is a Saccharomyces cerevisiae mitochondrial DEAD-box RNA helicase protein that is essential for efficient in vivo splicing of all group I and group II introns and for activation of mRNA translation. Catalysis of intron splicing by Mss116 is coupled to its ATPase activity. Knowledge of the kinetic pathway(s) and biochemical intermediates populated during RNA-stimulated Mss116 ATPase is fundamental for defining how Mss116 ATP utilization is linked to in vivo function. We therefore measured the rate and equilibrium constants underlying Mss116 ATP utilization and nucleotide-linked RNA binding. RNA accelerates the Mss116 steady-state ATPase ∼ 7-fold by promoting rate-limiting ATP hydrolysis such that inorganic phosphate (P_i) release becomes (partially) rate-limiting. RNA binding displays strong thermodynamic coupling to the chemical states of the Mss116-bound nucleotide such that Mss116 with bound ADP-P_i binds RNA more strongly than Mss116 with bound ADP or in the absence of nucleotide. The predominant biochemical intermediate populated during in vivo steady-state cycling is the strong RNA-binding Mss116-ADP-P_i state. Strong RNA binding allows Mss116 to fulfill its biological role in the stabilization of group II intron folding intermediates. ATPase cycling allows for transient population of the weak RNA-binding ADP state of Mss116 and linked dissociation from RNA, which is required for the final stages of intron folding. In cases where Mss116 functions as a helicase, the data collectively favor a model in which ATP hydrolysis promotes a weak-to-strong RNA binding transition that disrupts stable RNA duplexes. The subsequent strong-to-weak RNA binding transition associated with P_i release dissociates Mss116-RNA complexes, regenerating free Mss116.     

The pathway of inorganic-phosphate efflux from isolated liver mitochondria during adenosine triphosphate hydrolysis

1. The distribution of P_i between mitochondria and suspending medium during uncoupler-stimulated hydrolysis of ATP by rat liver mitochondria [Tyler (1969) Biochem. J. 111, 665–678] has been reinvestigated, by using either mersalyl or N-ethylmaleimide as inhibitors of P_i transport and either buffered sucrose/EDTA or LiCl/EGTA solutions as suspending medium. More than 75% of the total P_i liberated was retained in mitochondria treated with either inhibitor at all ATP concentrations tested (0.2–2.5mm). With low ATP concentrations and mersalyl-treated mitochondria incubated in sucrose/EDTA, virtually all the P_i liberated was retained in the mitochondria. 2. Larger amounts of P_i appeared in the suspending medium during ATPase activity, despite the presence of N-ethylmaleimide, when LiCl/EGTA was used as suspending medium compared with sucrose/EDTA. Two sources of this P_i were identified: (a) a slow efflux of P_i from mitochondria to suspending medium despite the presence of N-ethylmaleimide; (b) a slow ATPase activity insensitive to carboxyatractyloside, which was stimulated by added Mg²⁺, partially inhibited by oligomycin or efrapeptin and strongly inhibited by EDTA. 3. It is concluded that liver mitochondria preparations contain two distinct forms of ATPase activity. The major activity is associated with coupled mitochondria of controlled permeability to adenine nucleotides and P_i and is stimulated strongly by uncoupling agents. The minor activity is associated with mitochondria freely permeable to adenine nucleotides and P_i, is unaffected by uncoupling agents and is activated by endogenous or added Mg²⁺. 4. When mitochondria treated with mersalyl were incubated in buffered sucrose solution, almost all the P_i liberated was recovered in the suspending medium, unless inhibitors of P_i-induced large-amplitude swelling such as EDTA, EGTA, antimycin, rotenone, nupercaine or Mg²⁺ were added. Thus the loss of the specific permeability properties of the mitochondrial inner membrane associated with large-amplitude swelling also influences the extent of P_i retention during ATPase activity. 5. The results confirm the previous conclusion (Tyler, 1969) that the P_i transporter provides the sole pathway for P_i efflux during uncoupler-stimulated ATP hydrolysis by mitochondria. It is concluded that more recent hypotheses concerning the influence of Mg²⁺ on mersalyl inhibition of the P_i transporter [Siliprandi, Toninello, Zoccaroto & Bindoli (1975) FEBS Lett. 51, 15–17] and a postulated role of the adenine nucleotide exchange carrier in P_i efflux [Reynafarje & Lehninger (1978) Proc. Natl. Acad. Sci. U.S.A. 75, 4788–4792] are erroneous and should be discarded.     

Inhibition of the (Na+ + K+)-dependent ATPase by inorganic phosphate

Inhibition of the $\text{(}\text{Na}{}^{\mathrm{+}}\text{+ K}{}^{\mathrm{+}}\text{)-dependent}$ ATPase by inorganic phosphate, P_i, was examined in terms of product inhibition of the various activities catalyzed by an enzyme preparation from rat brain, and considered in terms of the specific transport processes of the membrane Na⁺,K⁺-pump that these activities reflect. The K⁺-dependent phosphatase activity of the enzyme was most sensitive to P_i, and inhibition was competitive toward the substrate, nitrophenyl phosphate, as would be expected if P_i were released from the same enzyme form that bound substrate. However, this enzymatic activity does not seem to represent a transport process, and thus a cyclical discharge of K⁺ may not be involved. The Na⁺-dependent exchange activity was unaffected by P_i, in accord with the absence of P_i release in the reaction sequence. For the corresponding Na⁺/Na⁺ exchange function of the pump, which reportedly does not involve ATP hydrolysis either, prior release of P_i obviously cannot be required for Na⁺ discharge. With the Na⁺-dependent ATPase activity, measured using micromolar concentrations of ATP, P_i inhibited, but far less than with the phosphatase activity, and inhibition was not competitive toward ATP. Moreover, inhibition decreased as the Na⁺ concentration was raised from 10 to 100 mM. This elevated concentration of Na⁺ also led to substrate inhibition. For this ATPase activity, and the corresponding transport process, uncoupled Na⁺ efflux, the findings suggest that Na⁺ discharge follows P_i release, in contrast to Na⁺/Na⁺ exchange. The $\text{(}\text{Na}{}^{\mathrm{+}}\text{+ K}{}^{\mathrm{+}}\text{)-dependent}$ ATPase activity, measured with millimolar concentrations of ATP and reflecting the coupled Na⁺,K⁺-transport function, was similarly sensitive to P_i, and again inhibition was not competitive toward ATP. However, in this case inhibition did not increase as the Na⁺ concentration was lowered. For this activity, and the associated transport process, the site of Na⁺ discharge in the overall reaction sequence remains unresolved.     

Pyridoxylation of essential lysine residues of mitochondrial adenosine triphosphatase

1. Soluble beef-heart mitochondrial ATPase (F₁) was incubated with [³H]pyridoxal 5′-phosphate and the Schiffbase complex formed was reduced with sodium borohydride. Spectral measurements indicate that lysine residues are modified and gel electrophoresis in the presence of detergent shows the tritium label to be associated with the two largest subunits, α and β. 2. In the absence of protecting ligands, the loss of ATP hydrolysis activity is linearly dependent on the level of pyridoxylation with complete inactivation correlating to 10 mol pyridoxamine phosphate incorporated per mol enzyme. Partial inactivation of F₁ with pyridoxal phosphate has no effect on either the K_m for ATP or the ability of bicarbonate to stimulate residual hydrolysis activity, suggesting a mixed population of fully active and fully inactive enzyme. 3. In the presence of excess magnesium, the addition of ADP or ATP, but not AMP, decreases the rate and extent of modification of F₁ by pyridoxal phosphate. The non-hydrolyzable ATP analog, 5′-adenylyl-β, γ-imidodiphosphate, is particularly effective in protecting F₁ against both modification and inactivation. Efrapeptin and P_i have no effect on the modification reaction. 4. Prior modification of F₁ with pyridoxal phosphate decreases the number of exchangeable nucleotide binding sites by one. However, pyridoxylation of F₁ is ineffective in displacing endogenous nucleotides bound at non-catalytic sites and does not affect the stoichiometry of P_i binding. 5. The ability of nucleotides to protect against modification and inactivation by pyridoxal phosphate and the loss of one exchangeable nucleotide site with the pyridoxylation of F₁ suggest the presence of a positively charged lysine residue at the catalytic site of an enzyme that binds two negatively charged substrates.     

Regulation of C4 photosynthesis: mechanism of activation and inactivation of extracted pyruvate, inorganic phosphate dikinase in relation to dark/light regulation

Requirements for activation of inactive pyruvate, inorganic phosphate (P_i) dikinase extracted from darkened maize leaves were examined. Incubation with P_i plus dithiothreitol resulted in a rapid recovery of activity comparable to that in illuminated leaves. However, contrary to previous findings, most of this activity (60–95%) was recovered by adding P_i alone. There was no activation with dithiothreitol alone. Dependency on dithiothreitol, in addition to P_i was minimal at about pH 7.5 but was substantial at higher pH. Anaerobic conditions did not enhance P_i-dependent activation. Active enzyme, isolated from illuminated leaves, was inactivated by incubating with ADP and this occurred in the presence of dithiothreitol. ATP and AMP were not effective but ATP may be a corequirment for ADP-dependent inactivation. Enzyme inactivated by ADP required P_i for reactivation. We conclude that interconversion of dithiol and disulfide forms of the enzyme is not critical for the dark/light regulation of pyruvate, P_i dikinase. The primary mechanism apparently involves an ADP-induced transformation to an inactive form which undergoes a P_i-mediated reactivation.     

Partial Purification and Characterization of the Phosphate Transporter from Bovine Heart Mitochondria

A highly active phosphate transporter was extracted with octylglucoside from bovine heart submitochondrial particles that were first partially depleted of other membrane components. It was then partially purified by ammonium sulfate fractionation. After reconstitution of the transporter into liposomes prepared with a crude mixture of soybean phospholipids, the P_i/OH exchange, but not the P_i/P_i exchange, was stimulated three- to fourfold by valinomycin and nigericin in the presence of K⁺. Both P_i/OH and P_i/P_i exchange activities were sensitive to mercurials and other SH reagents. The rutamycin-sensitive ATPase complex from mitochondria was reconstituted together with the phosphate transporter and adenine nucleotide transporter into liposomes. After inhibition of externally located ATPase, the hydrolysis of ATP was sensitive to atractyloside and mersalyl.     

Purification and reconstitution of the 32Pi-ATP exchange activity of bovine chromaffin granule membrane

Ghosts derived from bovine chromaffin granules have a ³²P_i-ATP exchange activity which is associated with the H⁺ pump of that membrane. This activity was low when compared to bacteria, chloroplasts or submitochondrial particles, but had similar properties (K_m for ATP and P_i, ATP/Mg²⁺ ratio, pH profile, inhibition by dicyclohexylcarbodiimide and tributyltin) to the ATPase from above membranes. The ³²P_i-ATP exchange activity was solubilized by cholate/octylglucoside mixtures. The soluble extract was lipid depleted by ammonium sulfate fractionation and partially purified by sucrose gradient centrifugation. The purified preparation was reconstituted with phospholipids by freeze-thawing. The reconstituted vesicles had a ³²P_i-ATP exchange sensitive to dicyclohexylcarbodiimide and trybutyltin and an ATPase with a sensitivity to the inhibitors which varied with the reconstitution conditions. The α- and β-subunits of F₁-ATPase were major components of the preparation.     

Tightly bound nucleotides of the energy-transducing ATPase,and their role in oxidative phosphorylation. I. The Paracoccus denitrificans system

1. The coupling ATPase of Paracoccus denitrificans can be removed from the membrane by washing coupled membrane fragments at low salt concentrations.2. This ATPase resembles coupling ATPases of mitochondria, chloroplasts and other bacteria. It is a negatively charged protein of molecular weight about 300 000. An inhibitor protein is bound tightly to the ATPase in vivo, and can be destroyed by trypsin treatment.3. ATP and ADP are found tightly bound to the coupling ATPase of P. denitrificans, both in its membrane-bound and isolated state. The ATP/ADP ratio on the enzyme is greater than one.4. Under de-energised conditions, the bound nucleotides are not available to the suspending medium. When the membrane is energised however, the bound nucleotides can exchange with added nucleotides and incorporate ³²P_i. ³²P_i is incorporated into the β and γ positions of the bound nucleotides, but β-labelling probably does not occur on the coupling ATPase.5. Uncouplers inhibit the exchange of the free nucleotides or ³²P_i into the bound nucleotides, while venturicidin (an energy transfer inhibitor) and aurovertin stimulate the exchange.6. The response of the bound nucleotides to energisation is consistent with their being involved directly in the mechanism of oxidative phosphorylation.     

Properties of a plasmalemma ATPase of the maize scutellum

Properties of a plasmalemma phosphatase of the maize scutellum, tentatively identified as an ATPase in a previous paper, were investigated. Fresh and frozen-thawed scutellum slices, that had been treated with 10 mM HCl to destroy acid phosphatases, were used as a source of enzyme. With the exceptions of the Na⁺, K⁺ and dinitrophenol experiments, the two kinds of slices gave similar results. ATP and CTP were the best substrates for the enzyme followed by TTP, UTP, CDP, ADP and GTP. UDP, nucleoside monophosphates, sugar phosphates, inorganic pyrophosphate and p-nitrophenyl phosphate were relatively ineffective as substrates. The K_m's for ATP and ADP were 0.65 and 5 mM, respectively, but the two substrates gave the same V_max (49.8 μmol P_i/hr/g slices). Previously, it was shown that the products of ATP hydrolysis are ADP, AMP and P_i. Using these previous results and from the time courses of ATP disappearance from the bathing solution and the appearance of P_i and ADP, it was concluded that ATP and ADP were hydrolysed by the same enzyme. The ATPase was not inhibited by oligomycin. N-N′-Dicyclohexylcarbodiimide (DCCD) was a poor inhibitor, and a water soluble analog of DCCD, 1-ethyl-3 (3 dimethyl-aminopropyl)-carbodiimide, gave only 33% inhibition. The relative effectiveness of divalent cations for stimulating ATPase activity was Mn²⁺ > Mg²⁺ ? Ca²⁺ > Co²⁺ · Na⁺ and K⁺ gave a small additional stimulation in the presence of Mg²⁺. However, Na⁺ and K⁺ gave a much greater stimulation when no divalent cation was added, and this occurred only when fresh slices were used. Dinitrophenol also increased ATPase activity only when fresh slices were used. Since it is likely that both the uptake of Na⁺ and K⁺ and the action of dinitrophenol would lower the electrochemical gradient of protons across the plasmalemma, the different results obtained with fresh slices indicate that the ATPase in these slices was under the constraint of a proton gradient.     

The ATPase cycle mechanism of the DEAD-box rRNA helicase, DbpA

DEAD-box proteins are ATPase enzymes that destabilize and unwind duplex RNA. Quantitative knowledge of the ATPase cycle parameters is critical for developing models of helicase activity. However, limited information regarding the rate and equilibrium constants defining the ATPase cycle of RNA helicases is available, including the distribution of populated biochemical intermediates, the catalytic step(s) that limits the enzymatic reaction cycle, and how ATP utilization and RNA interactions are linked. We present a quantitative kinetic and equilibrium characterization of the ribosomal RNA (rRNA)-activated ATPase cycle mechanism of DbpA, a DEAD-box rRNA helicase implicated in ribosome biogenesis. rRNA activates the ATPase activity of DbpA by promoting a conformational change after ATP binding that is associated with hydrolysis. Chemical cleavage of bound ATP is reversible and occurs via a γ-phosphate attack mechanism. ADP-P_i and RNA binding display strong thermodynamic coupling, which causes DbpA-ADP-P_i to bind rRNA with > 10-fold higher affinity than with bound ATP, ADP or in the absence of nucleotide. The rRNA-activated steady-state ATPase cycle of DbpA is limited both by ATP hydrolysis and by P_i release, which occur with comparable rates. Consequently, the predominantly populated biochemical states during steady-state cycling are the ATP- and ADP-P_i-bound intermediates. Thermodynamic linkage analysis of the ATPase cycle transitions favors a model in which rRNA duplex destabilization is linked to strong rRNA and nucleotide binding. The presented analysis of the DbpA ATPase cycle reaction mechanism provides a rigorous kinetic and thermodynamic foundation for developing testable hypotheses regarding the functions and molecular mechanisms of DEAD-box helicases.     

The ATPase Cycle of PcrA Helicase and Its Coupling to Translocation on DNA

The superfamily 1 bacterial helicase PcrA has a role in the replication of certain plasmids, acting with the initiator protein (RepD) that binds to and nicks the double-stranded origin of replication. PcrA also translocates single-stranded DNA with discrete steps of one base per ATP hydrolyzed. Individual rate constants have been determined for the DNA helicase PcrA ATPase cycle when bound to either single-stranded DNA or a double-stranded DNA junction that also has RepD bound. The fluorescent ATP analogue 2′(3′)-O-(N-methylanthraniloyl)ATP was used throughout all experiments to provide a complete ATPase cycle for a single nucleotide species. Fluorescence intensity and anisotropy stopped-flow measurements were used to determine rate constants for binding and release. Quenched-flow measurements provided the kinetics of the hydrolytic cleavage step. The fluorescent phosphate sensor MDCC-PBP was used to measure phosphate release kinetics. The chemical cleavage step is the rate-limiting step in the cycle and is essentially irreversible and would result in the bound ATP complex being a major component at steady state. This cleavage step is greatly accelerated by bound DNA, producing the high activation of this protein compared to the protein alone. The data suggest the possibility that ADP is released in two steps, which would result in bound ADP also being a major intermediate, with bound ADP·P_i being a very small component. It therefore seems likely that the major transition in structure occurs during the cleavage step, rather than P_i release. ATP rebinding could then cause reversal of this structural transition. The kinetic mechanism of the PcrA ATPase cycle is very little changed by potential binding to RepD, supporting the idea that RepD increases the processivity of PcrA by increasing affinity to DNA rather than affecting the enzymatic properties per se.     

Rapid and efficient method for analyzing phosphorylation of the S6 ribosomal protein in 32Pi-labeled,tissue culture cells

We describe a method for studying the phosphorylation of the S6 ribosomal protein in intact cells. The procedure has the advantage of using few cells, little ³²P_i, and by using an air-driven centrifuge, many samples can be processed in a short time. Metabolically labeling the ribosomes with [³H]uridine before the experiment provides a measure of ribosome yield. The amount of ³²P_i incorporated into proteins other than S6, which cosediment with the ribosomes, increases by the same amount as the specific activity of [³²P]ATP increases, when the cells are stimulated by prostaglandin F_2α, insulin, epidermal, or fibroblast growth factor, or serum; whereas the ³²P_i incorporated into S6 increases by a factor greater than the increase in the specific activity of [³²P]ATP. We show that the phosphate on S6 turns over at least as rapidly as does the phosphate on ATP. This last observation allows us to use a procedure, which we have outlined for determining the absolute amount of phosphate added to S6 due to a stimulus.     

Polyphosphate Kinase of Acinetobacter sp. Strain ADP1: Purification and Characterization of the Enzyme and Its Role during Changes in Extracellular Phosphate Levels

Polyphosphate (polyP) is a ubiquitous biopolymer whose function and metabolism are incompletely understood. The polyphosphate kinase (PPK) of Acinetobacter sp. strain ADP1, an organism that accumulates large amounts of polyP, was purified to homogeneity and characterized. This enzyme, which adds the terminal phosphate from ATP to a growing chain of polyP, is a 79-kDa monomer. PPK is sensitive to magnesium concentrations, and optimum activity occurs in the presence of 3 mM MgCl₂. The optimum pH was between pH 7 and 8, and significant reductions in activity occurred at lower pH values. The greatest activity occurred at 40°C. The half-saturation ATP concentration for PPK was 1 mM, and the maximum PPK activity was 28 nmol of polyP monomers per μg of protein per min. PPK was the primary, although not the sole, enzyme responsible for the production of polyP in Acinetobacter sp. strain ADP1. Under low-phosphate (P_i) conditions, despite strong induction of the ppk gene, there was a decline in net polyP synthesis activity and there were near-zero levels of polyP in Acinetobacter sp. strain ADP1. Once excess phosphate was added to the P_i-starved culture, both the polyP synthesis activity and the levels of polyP rose sharply. Increases in polyP-degrading activity, which appeared to be mainly due to a polyphosphatase and not to PPK working in reverse, were detected in cultures grown under low-P_i conditions. This activity declined when phosphate was added.     

Involvement of phosphate-modified ATP analogs in the reactions of oxidative phosphorylation

Various analogs of adenosine 5′-triphosphate with a modified terminal phosphate group have been tested in energy-requiring reactions with intact mitochondria and submitochondrial particles.It is shown that the fluorophosphate analog ATP(γF) is a strong inhibitor of mitochondrial respiration and of energy requiring reactions which involve the participation of high energy intermediates, generated aerobically by the respiratory chain. On the other hand, ATP(γF) does not affect the ATPase activity of intact or disrupted mitochondria and is less effective in inhibiting ATP-driven reactions.The imidophosphate analog AMP-P(NH)P also inhibits the partial reactions of oxidative phosphorylation, but does not affect ATP synthesis from ADP and P_i. In contrast to ATP(γF), it is a strong inhibitor of both soluble and membrane-bound mitochondrial ATPases.The biological implication of the complementary effects of ATP(γF) and AMP-P(NH)P on mitochondria-catalysed reactions is discussed while suggesting the use of such nucleotide analogs as specific tools for the study of ATP-forming and ATP-utilizing reactions in mitochondria.     

The yeast mitochondrial adenosine triphosphatase complex. Purification, subunit composition, and some effects of protease inhibitors.

(i) The method of preparing the oligomycin-insensitive F₁-ATPase by chloroform treatment of mitochondrial membranes (Beechey et al., 1975, Biochem. J.148, 533–537) has been modified such that a five-subunit protein is obtained from yeast with an activity of 140 μmol of ATP hydrolyzed/min/mg of protein. Repetition of this procedure in the presence of protease inhibitors (in particular, p-aminobenzamidine) allows isolation of a four-subunit protein with an activity of 243 μmol of ATP hydrolyzed/min/ mg of protein, (ii) A modified procedure is described for the preparation of the yeast oligomycin-sensitive F₁-F₀ ATPase complex, making use of protease inhibitors throughout and solubilization of the ATPase from mitochondrial membranes using Triton X-100 and sodium deoxycholate simultaneously. Two polypeptides Of 42,000 and 29,000 molecular weight are eliminated, the largest corresponding to the missing band of the F₁ sector. The complex retains oligomycin- and uncoupler-sensitive ATP-³²P_i exchange and ATP-driven proton uptake, indicating the retention of a complete coupling mechanism. (iii) F₁-ATPase is released from the F₁-F₀ complex by brief heating at 50 °C in the presence of ATP. The remaining hydrophobic polypeptides aggregate and are isolated by centrifugation. The F₁ sector can be isolated containing either four or five subunits depending on whether the starting F₁-F₀ complex contained the 42,000 and 29,000 molecular weight polypeptides. (iv) Sensitivity of the F₁-F₀ ATPase complex to oligomycin and dicyclohexylcarbodiimide varies considerably depending on the activity measured and whether the complex was first reconstituted with phospholipids. The degree of inhibitor sensitivity is considered a poor guide to intactness of the complex.     

Requirement of medium ADP for the steady-state hydrolysis of ATP by the proton-translocating Paracoccus denitrificans Fo·F1-ATP synthase

F_o·F₁-ATP synthase in inside-out coupled vesicles derived from Paracoccus denitrificans catalyzes P_i-dependent proton-translocating ATPase reaction if exposed to prior energization that relieves ADP·Mg²⁺-induced inhibition (Zharova, T.V. and Vinogradov, A.D. (2004) J. Biol. Chem.,279, 12319–12324). Here we present evidence that the presence of medium ADP is required for the steady-state energetically self-sustained coupled ATP hydrolysis. The initial rapid ATPase activity is declined to a certain level if the reaction proceeds in the presence of the ADP-consuming, ATP-regenerating system (pyruvate kinase/phosphoenol pyruvate). The rate and extent of the enzyme de-activation are inversely proportional to the steady-state ADP concentration, which is altered by various amounts of pyruvate kinase at constant ATPase level. The half-maximal rate of stationary ATP hydrolysis is reached at an ADP concentration of 8 × 10⁻⁶ M. The kinetic scheme is proposed explaining the requirement of the reaction products (ADP and P_i), the substrates of ATP synthesis, in the medium for proton-translocating ATP hydrolysis by P. denitrificans F_o·F₁-ATP synthase.     

Interaction of homogeneous mitochondrial ATPase from rat liver with adenine nucleotides and inorganic phosphate

Mitochondrial ATPase from rat liver mitochondria contains multiple nucleotide binding sites. At low concentrations ADP binds with high affinity (1 mole/mole ATPase, K_D = 1–2 μM). At high concentrations, ADP inhibits ATP hydrolysis presumably by competing with ATP for the active site (K_I = 240–300 μM). As isolated, mitochondrial ATPase contains between 0.6 and 2.5 moles ATP/mole ATPase. This “tightly bound” ATP can be removed by repeated precipitations with ammonium sulfate without altering hydrolytic activity of the enzyme. However, the ATP-depleted enzyme must be redissolved in high concentrations of phosphate to retain activity. AMP-PNP (adenylyl imidodiphosphate) replaces tightly bound ATP removed from the enzyme and inhibits ATP hydrolysis. AMP-PNP has little effect on high affinity binding of ADP. Kinetic studies of ATP hydrolysis reveal hyperbolic velocity vs. ATP plots, provided assays are done in bicarbonate buffer or buffers containing high concentrations of phosphate. Taken together, these studies indicate that sites on the enzyme not directly associated with ATP hydrolysis bind ATP or ADP, and that in the absence of bound nucleotide, P_i can maintain the active form of the enzyme.     

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.     

ATP binding and hydrolysis steps of the uni-site catalysis by the mitochondrial F1-ATPase are affected by inorganic phosphate

The effect of inorganic phosphate (P_i) on uni-site ATP binding and hydrolysis by the nucleotide-depleted F₁-ATPase from beef heart mitochondria (ndMF₁) has been investigated. It is shown for the first time that P_i decreases the apparent rate constant of uni-site ATP binding by ndMF₁ 3-fold with the K_d of 0.38 ± 0.14 mM. During uni-site ATP hydrolysis, P_i also shifts equilibrium between bound ATP and ADP + P_i in the direction of ATP synthesis with the K_d of 0.17 ± 0.03 mM. However, 10 mM P_i does not significantly affect ATP binding during multi-site catalysis.