An inhibitory high affinity binding site for ADP in the oligomycin-sensitive ATPase of beef heart submitochondrial particles.

Kinetic evidence are presented for the existence of a high affinity inhibitory site for ADP /K_i < 10^?7 M/ in the oligomycin-sensitive ATPase of beef heart submitochondrial particles. The ATPase·ADP complex is completely inactive in the ATPase reaction; it can be converted into active ATPase in a slow ATP-dependent reaction. The dependence of a first order rate constant for activation of the enzyme·ADP complex on concentration of ATP gives a K_m value equal to that for ATP in the ATPase reaction. The data obtained suggest that the membrane-bound ATPase complex contains two kinetically distinct nucleotide-binding centers, i.e. center 1 binds ATP or ADP with a formation of enzyme-substrate or enzyme-competitive inhibitor complexes: center 2 binds ADP with a formation of a complex which is able to bind ATP in center 1 and unable to hydrolyze the bound ATP. The binding of ATP or ADP in center 1 changes the reactivity of center 2 towards ADP.     

Inhibition of photophosphorylation by ATP and the role of magnesium in photophosphorylation

ATP and pyrophosphate at high concentration (> 1 mM) inhibited photophosphorylation of isolated spinach chloroplasts in the normal salt medium and did not cause stimulation of electron transport. The inhibition of photophosphorylation by ATP or pyrophosphate was shown to be abolished by the addition of excess MgCl₂, ADP and phosphate. It has been demonstrated that the rates of photophosphorylation in the absence and presence of ATP or pyrophosphate are determined similarly by the concentrations of magnesium-ADP (Mg · ADP^?) and magnesium-phosphate (Mg · P_i) complexes.It is highly probable that Mg · ADP^? and Mg · P_i, but not free ADP and free phosphate, are the active form of the substrates of photophosphorylation. This is in support of the view that ATP inhibits photophosphorylation by decreasing the concentration of Mg²⁺ which is available for the formation of the complex with ADP and phosphate.     

Studies on photophosphorylation utilizing methylene diphosphonate analogs of ADP and ATP

Spinach chloroplasts were able to photophosphorylate the ADP analog α,β-methylene adenosine 5′-diphosphate (AOPCP). Phosphorylation of AOPCP was catalyzed by chloroplasts that were washed or dialyzed to remove free endogenous nucleotides. In the presence of glucose, hexokinase, AOPCP and ³²P_i, the ³²P label was incorporated into α,β-methylene adenosine 5′-triphosphate (AOPCPOP).In contrast to photophosphorylation of AOPCP, the ATP analog AOPCPOP was a poor substrate for the ATP-P_i exchange reaction and its hydrolysis was neither stimulated by light and dithiothreitol nor inhibited by Dio-9.Photophosphorylation of AOPCP was inhibited by the α,β- and β,γ-substituted methylene analogs of ATP, while phosphorylation of ADP was unaffected by them. The ATP-P_i exchange was also unaffected by both ATP analogs, while the weak AOPCPOP-P_i exchange was inhibited by the β,γ-methylene analog of ATP.Direct interaction of methylene analogs with the chloroplast coupling factor ATPase was indicated by the enzymatic hydrolysis of AOPCPOP on polyacrylamide gels.     

The stereochemical course of thiophosphoryl group transfer catalyzed by adenosine kinase

Adenosine kinase was partially purified form beef liver and used to catalyze the conversion of (γR)ATPγS,γ¹⁸O and adenosine to ADP and AMPαS,α¹⁸O. The configuration at phosphorus in AMPαS,α¹⁸O was established by subjecting it to stereospecific phosphorylation to (αS)ATPαS,α¹⁸O and showing that only the nonbridging oxygen bonded to the α-P was enriched with ¹⁸O. The configuration at α-P in AMPαS,α¹⁸O was therefore S, and the transfer of the [¹⁸O]thiophosphoryl group occurred with $\text{inversion}$ of configuration.     

Magnesium spin state determines the energetics of an adenosinetriphosphate molecule

The molecular dynamics method (density functional theory) DFT:B3LYP (6-3IG** basis set, t = 310 K) was used to study interactions between a molecule of adenosinetriphosphate (ATP) (ATP subsystem) and the [Mg(H₂O)₆]²⁺ magnesium cofactor (Mg subsystem) in an aqueous medium simulated by 78 water molecules in the singlet (S) and triplet (T) states. Potential energy surfaces (PESs) for the S (lowest in energy) and T states (highest in energy) are significantly separated in space. Motion along them directs the Mg complex either to oxygen atoms of the γ-β-phosphate groups (O1–O2) (S state of PES) or to oxygen atoms of the β-α-phosphate groups (O2–O3) (T state of PES). Chelation of the γ-β- and β-α-phosphates leads to formation of a stable low-energy ([Mg(H₂O)₄-(OI-O2)ATP]^2?) complex or a metastable high-energy ([Mg(H₂O)₂-(O2–O3)ATP]^2?) complex, respectively, which differ in number of water molecules surrounding the Mg atom. Intersection of two T PESs is accompanied by formation of an unstable state characterized by redistribution of spins between the Mg and ATP subsystems. This state, being sensitive to interaction with the Mg nuclear spin (²⁵Mg), induces an unpaired electron spin, which initiates the ATP cleavage by the ion-radical mechanism, yielding a reactive ion radical of adenosinemonophosphate (·AMP^?), which was earlier found experimentally by the method of chemically induced dynamic nuclear polarization (CIDNP). Biological aspects of the results obtained are discussed.     

AMPK modulates glucose-sensing in insulin-secreting cells by altered phosphotransfer to KATP channels

Glucose-sensing (GS) behaviour in pancreatic β-cells is dependent on ATP-sensitive K⁺ channel (K_ATP) activity, which is controlled by the relative levels of the K_ATP ligands ATP and ADP, responsible for closing and opening K_ATP, respectively. However, the mechanism by which β-cells transfer energy status from mitochondria to K_ATP, and hence to altered electrical excitability and insulin secretion, is presently unclear. Recent work has demonstrated a critical role for AMP-activated protein kinase (AMPK) in GS behaviour of cells. Electrophysiological recordings, coupled with measurements of gene and protein expression were made from rat insulinoma cells to investigate whether AMPK activity regulates this energy transfer process. Using the whole-cell recording configuration with sufficient intracellular ATP to keep K_ATP closed, raised AMPK activity induced GS electrical behaviour. This effect was prevented by the AMPK inhibitor, compound C and required a phosphotransfer process. Indeed, high levels of intracellular phosphocreatine or the presence of the adenylate kinase (AK) inhibitor AP₅A blocked this action of AMPK. Using conditions that maximised AMPK-induced K_ATP opening, there was a significant increase in AK1, AK2 and UCP2 mRNA expression. Thus we propose that K_ATP opening in response to lowered glucose concentration requires AMPK activity, perhaps in concert with increased AK and UCP2 to enable mitochondrial-derived ADP signals to be transferred to plasma membrane K_ATP by phosphotransfer cascades.     

Relationships of inosine triphosphate and bicarbonate effects on F1 ATPase to the binding change mechanism

Two interesting previously reported properties of mitochondrial F₁ ATPase have been confirmed and have been examined by¹⁸O exchange measurements to assess if they are consistent with sequential participation of catalytic sites during ATP hydrolysis. These are the ability of HCO ₃ ^– to increase reaction rate with apparent loss of cooperative interaction between subunits and the ability of ITP to accelerate the hydrolysis of a low concentration of ATP. The effect of HCO ₃ ^– was tested at concentrations of ATP lower than previous measurements. The activation disappeared when ATP was reduced to 0.1 µM. The HCO ₃ ^– activation at higher ATP concentrations did not change the extent of reversal of the cleavage of tightly bound ATP at the catalytic site, as measured by the average number of water oxygens incorporated with each P_i formed when 5 or 10 µM ATP is hydrolyzed. The data are consistent with sequential site participation with HCO ₃ ^– acceleration of ADP departure after a binding change that stops¹⁸O exchange and loosens ADP binding.When ITP concentration was lowered during net ITP hydrolysis by F₁ ATPase an increase in water oxygen incorporation into P_i formed is observed, as noted previously for ATP hydrolysis. The acceleration of the cleavage of a constant low concentration of [-¹⁸O]ATP by concomitant hydrolysis of increasing concentrations of ITP was accompanied by a decrease in water oxygen incorporation with each P_i formed from the ATP. These results add to evidence for the binding change mechanism for F₁ ATPase with sequential participation of catalytic sites.     

The stoichiometry (ATP/2e− ratio) of non-cyclic photophosphorylation in isolated spinach chloroplasts

1. The stoichiometry of non-cyclic photophosphorylation and electron transport in isolated chloroplasts has been re-investigated. Variations in the isolation and assay techniques were studied in detail in order to obtain optimum conditions necessary for reproducibly higher ADP/O (equivalent to ATP/2e^?) and photosynthetic control ratios.2. Studies which we carried out on the possible contribution of cyclic phosphorylation to non-cyclic phosphorylation suggested that not more than 10% of the total phosphorylation found could be due to cyclic phosphorylation.3. Photosynthetic control, and the uncoupling of electron transport in the presence of NH₄Cl, were demonstrated using oxidised diaminodurene as the electron acceptor. A halving of the ADP/O ratio was found, suggesting that electrons were being accepted between two sites of energy conservation, one of which is associated with Photosystem I and the other associated with Photosystem II.4. ATP was shown to inhibit State 2 and State 3 of electron transport, but not State 4 electron transport or the overall ADP/O ratio, thus confirming its activity as an energy transfer inhibitor. It is suggested that part of the non-phosphorylating electron transport rate (State 2) which is not inhibited by ATP is incapable of being coupled to subsequent phosphorylation triggered by the addition of ADP (State 3). If the ATP-insensitive State 2 electron transport is deducted from the State 3 electron transport when calculating the ADP/O ratio, a value of 2.0 is obtained.5. The experiments reported demonstrate that there are two sites of energy conservation in the non-cyclic electron transfer pathway: one associated with Photosystem II and the other with Photosystem I. Thus, non-cyclic photophosphorylation can probably produce sufficient ATP and NADPH “in vivo” to allow CO₂ fixation to proceed.     

Substrate specificity of a recombinant d‐lyxose isomerase from Serratia proteamaculans that produces d‐lyxose and d‐mannose

Aims: Characterization of substrate specificity of a d ‐lyxose isomerase from Serratia proteamaculans and application of the enzyme in the production of d ‐lyxose and d ‐mannose. Methods and Results: The concentrations of monosaccharides were determined using a Bio‐LC system. The activity of the recombinant protein from Ser. proteamaculans was the highest for d ‐lyxose among aldoses, indicating that it is a d‐ lyxose isomerase. The native recombinant enzyme existed as a 54‐kDa dimer, and the maximal activity for d‐ lyxose isomerization was observed at pH 7·5 and 40°C in the presence of 1 mmol l^?1 Mn²⁺. The K_m values for d ‐lyxose, d ‐mannose, d ‐xylulose, and d ‐fructose were 13·3, 32·2, 3·83, and 19·4 mmol l^?1, respectively. In 2 ml of reaction volume at pH 7·5 and 35°C, d ‐lyxose was produced at 35% (w/v) from 50% (w/v) d ‐xylulose by the d‐ lyxose isomerase in 3 h, while d ‐mannose were produced at 10% (w/v) from 50% (w/v) d ‐fructose in 5 h. Conclusions: We identified the putative sugar isomerase from Ser. proteamaculans as a d ‐lyxose isomerase. The enzyme exhibited isomerization activity for aldose substrates with the C2 and C3 hydroxyl groups in the left‐hand configuration. High production rates of d‐ lyxose and d ‐mannose by the enzyme were obtained. Significance and Impact of the Study: A new d‐ lyxose isomerase was found, and this enzyme had higher activity for d ‐lyxose and d ‐mannose than previously reported enzymes. Thus, the enzyme can be applied in industrial production of d ‐lyxose and d ‐mannose.     

Phosphate- N base hydrogen bonds involving proton transfer with reference to the non-enzymic hydrolysis of ATP

IR spectra of aqueous solutions of 1:1 mixtures of H₂PO₄^? and various N bases have been studied as models for (POH?N) → (P^?O?H⁺N) hydrogen bonds. 50% proton transfer is observed when the pKa of the protonated N base is 1.1 smaller than that of the phosphate group. The hydrogen bonds are easily polarizable near this equilibrium. These results strongly support the conclusion that such bonds contribute 1) to the self-association of ATP and ADP and 2) to the association of the hydrolysis products ADP and inorganic phosphate.     

Phosphofructokinase of Solanum tuberosum tuber

Potato tuber phosphofructokinase was purified 19·.6-fold by a combination of ethanol fractionation and DEAE-cellulose column chromatography. The enzyme was very unstable; its pH optimum was 8·0. K_m for fructose-6-phosphate, ATP and Mg²⁺ was 2·1 × 10^?4 M, 4·5 × 10^?5 M and 4·0 × 10^?4 M respectively. ITP, GTP, UTP and CTP can act as phosphate donors, but are less active than ATP. Inhibition of enzyme activity by high levels of ATP was reversed by increasing the concentration of fructose-6-phosphate; the affinity of enzyme for fructose-6-phosphate decreased with increasing concentration of ATP. 5′-AMP, 3′,5′-AMP, 3′-AMP, deoxy AMP, UMP, IMP, CMP, GMP, ADP, CDP, GDP and UDP did not reverse the inhibition of enzyme by ATP. ADP, phosphoenolpyruvate and citrate inhibited phosphofructokinase activity but Pi did not affect it. Phosphofructokinase was not reactivated reversibly by mild change of pH and addition of effectors.     

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.     

Sequential hydrolysis of the γ- and β-phosphate groups of ATP by the ATP diphosphohydrolase from pig pancreas

The ATP dipbosphohydrolase (EC 3.6.1.5) from pig pancreas hydrolyzes triphospho- and diphosphonucleosides. The reaction products of ATP hydrolysis are ADP, AMP and orthophosphate, but AMP accumulates at a faster rate than ADP. A time-course study showed a simultaneous breakdown of ATP and ADP with initial rates for ATP and ADP hydrolysis of 2.1 and 3.8μmol/min per mg protein, respectively. However, the rates reached similar values toward the end of the incubation period. According to double reciprocal plots and Dixon plots, the K_m values for ATP and ADP are similar, V_max for ADP hydrolysis is twice the V_max for ATP hydrolysis and both nucleotides are competitive inhibitors of the other with their K_i values similar to their K_m. These results are consistent with a sequential hydrolysis of the two diphosphoester bonds of ATP: ATP first binds to the enzyme, its γ-phosphate group is hydrolyzed and released, resulting in an enzyme-ADP complex which either breaks down to free enzyme and ADP or is further processed via hydrolysis of the β-phosphate group, releasing free enzyme, AMP and P_i. The experimental data showed that the processing step is favored.     

MEMBRANE THIAMINE TRTPHOSPHATASE FROM RAT BRAIN: INHIBITION BY ATP AND ADP

—The hydrolysis of ThTP by rat brain membrane-bound ThTPase is inhibited by nucleoside diphosphates and triphosphates. ATP and ADP are most effective, reducing hydrolysis by 50% at concentrations of 2 × 10^?5m and 7·5 × 10^?5m respectively. Nucleoside monophosphates and free nuclcosides as well as P_i have no effect on enzyme activity. ThMP and ThDP also fail to inhibit hydrolysis in concentrations up to 5 × 10^?3m . Non-hydrolysable methylene phosphate analogs of ATP and ADP were used in further kinetic studies with the ThTPase. The mechanism of inhibition by these analogs is shown to be of mixed non-competitive nature for both compounds. An observed K_i, of 4 × 10^?5m for the ATP analog adenosine-PPCP and 9 × 10^?5m for the ADP analog adenosine-PCP is calculated at pH 6·5. Formation of the true enzyme substrate, the [Mg²⁺. ThTP] complex, is not significantly affected by concentrations of analogs producing maximal (>95%) inhibition of enzyme activity. Likewise the relationships between pH and observed K_m and pH and V_max are not shifted by the presence of similar concentrations of inhibitor.     

H-ATPase Activity from Storage Tissue of Beta vulgaris: III. Modulation of ATPase Activity by Reaction Substrates and Products

Two distinct membrane fractions containing H⁺-ATPase activity were prepared from red beet. One fraction contained a H⁺-ATPase activity that was inhibited by NO₃⁻ while the other contained a H⁺-ATPase inhibited by vanadate. We have previously proposed that these H⁺-ATPases are associated with tonoplast (NO₃⁻-sensitive) and plasma membrane (vanadate-sensitive), respectively. Both ATPase were examined to determine to what extent their activity was influenced by variations in the concentration of ATPase substrates and products. The substrate for both ATPase was MgATP²⁻, and Mg²⁺ concentrations in excess of ATP had only a slight inhibitory effect on either ATPase. Both ATPases were inhibited by free ATP (i.e. ATP concentrations in excess of Mg²⁺) and ADP but not by AMP. The plasma membrane ATPase was more sensitive than the tonoplast ATPase to free ATP and the tonoplast ATPase was more sensitive than the plasma membrane ATPase to ADP.

Inhibition of both ATPases by free ATP was complex. Inhibition of the plasma membrane ATPase by ADP was competitive whereas the tonoplast ATPase demonstrated a sigmoidal dependence on MgATP²⁻ in the presence of ADP. Inorganic phosphate moderately inhibited both ATPases in a noncompetitive manner.

Calcium inhibited the plasma membrane but not the tonoplast ATPase, apparently by a direct interaction with the ATPase rather than by disrupting the MgATP²⁻ complex.

The sensitivity of both ATPases to ADP suggests that under conditions of restricted energy supply H⁺-ATPase activity may be reduced by increases in ADP levels rather than by decreases in ATP levels per se. The sensitivity of both ATPases to ADP and free ATP suggests that modulation of cytoplasmic Mg²⁺ could modulate ATPase activity at both the tonoplast and plasma membrane.

     

The crystal structure of a lyxose-bridged dimolybdate: a redetermination of the first monosaccharide-metal complex’s structure

The crystal structure of the first monosaccharide-metal complex ever reported, a β-d-lyxofuranose-bridged dimolybdate [Taylor, G. E.; Waters, J. M. Tetrahedron Lett.1981, 22, 1277-1278] was re-determined since the original work shows unbalanced charges, and thus an unclear protonation state of the lyxose ligand. As a result, the complex was found to be a monoanion whose charge is balanced by an ammonium counterion. The lyxose O5 atom is non-deprotonated, and the carbohydrate ligand is trianionic due to deprotonation of the O1, O2 and O3 hydroxy functions.     

The effects of ADP on reverse electron flow and the oxygen exchange reactions catalyzed by bovine heart muscle submitochondrial particles

1,N⁶-Ethenoadenosine diphosphate (ϵ-ADP) inhibits reverse electron flow (succinate → NAD⁺ driven by ATP) by competing with ATP, in contrast to ADP which we have shown previously to be a noncompetitive inhibitor. From these and other data it is concluded that the noncompetitive inhibition noted with ADP results from a combination of competitive inhibition plus non- or uncompetitive inhibition, the former occuring at a relatively nonspecific catalytic site and the latter at an extracatalytic site apparently quite specific for ADP. ADP, which stimulates ATP ⇌ H₂O and P_i ⇌ H₂O exchanges appears to be necessary for inhibition by arsenate of these exchanges. It is suggested that the ATP-supported P_i ⇌ H₂O exchange may be predominantly of the medium or intermediate type, depending on the concentrations of the Mg²⁺ complexes of ADP and P_i. Thus only exchanges involving medium ADP and P_i would be expected to show arsenate sensitivity.     

The Coupling of Electron Flow to ATP Synthesis in Pea and Maize Mesophyll Chloroplasts : I. INTERACTION OF ADENINE NUCLEOTIDES AND ENERGY TRANSFER INHIBITORS WITH THE COUPLING FACTOR COMPLEX

The rate of nonphosphorylating electron transport (in the absence of ADP and inorganic phosphate) in well-coupled (ATP/2e⁻ = 0.9-1.1) maize mesophyll chloroplasts is not modulated by external pH (6.5-8.5), low levels of ADP or ATP, or energy transfer inhibitors, e.g. triphenyltin and Hg²⁺ ions. In contrast nonphosphorylating electron flow in pea chloroplasts is sensitive to alterations in medium pH, and to the presence of adenine nucleotides and energy transfer inhibitors in the assay medium. Although ATP is without effect on the rate of basal electron transport in maize chloroplasts, steady-state proton uptake is stimulated 3- to 5-fold by low levels of ATP. These results suggest that differences may exist in the manner in which the coupling factor complex controls proton efflux from the intrathylakoid space in C₃ and C₄ mesophyll chloroplasts.     

Inhibition of Escherichia coli glutamine synthetase by phosphinothricin

The inhibition of Escherichia coli glutamine synthetase by phosphinothricin [2-amino-4-(methylphosphinyl)butanoic acid] has been studied. This amino acid was observed to function as an active site directed inhibitor exhibiting time-dependent inhibition of glutamine synthetase in the presence of ATP or adenylylimidodiphosphate (AMPPNP) but not adenylyl(β,γ-methylene) diphosphonate (AMPPCP). The inactivation was observed to be pseudo-first order. Phosphinothricin was also found to inhibit the enzyme reversibly under initial rate conditions and was competitive with respect to glutamate with K_1S = 18 ± 3 μm. The inactive enzyme inhibitor complex was found to contain approximately 11 molecules of ADP and of ³²P per dodecamer using [γ-³²P]ATP. Reactivation of the inactive enzyme complex was achieved by incubating the enzyme complex in 50 mm acetate (pH 4.4), 1 m KCl, and 0.40 m (NH₄)₂SO₄. ADP, phosphinothricin, and P_i were released upon reactivation.