Actin polymerization and ATP hydrolysis

This paper surveys several aspects of the consequences of ATP hydrolysis associated with actin polymerization, and their physiological implications. ATP hydrolysis occurs on F-actin in two subsequent reactions, cleavage of ATP followed by the slower release of P_i. The latter reaction is linked to a conformation change of the actin subunit that causes a destabilization of the actin-actin interactions in the filament, i.e., a structural change of the filament. The nature of the nucleotide bound to terminal subunits therefore affects the dynamics of actin filaments. It is shown that this regulation is different at the two ends, terminal F-ADP-P_i subunits being present at steady state at the barbed end, while F-ADP-subunits are present at the pointed end. While cleavage of ATP on F-actin is irreversible, P_i release is reversible, which allows the regulation of filament dynamics by cellular P_i. The nature of the divalent metal ion — Ca²⁺ or Mg²⁺ — tightly bound to actin, in direct interaction with ATP, also affects the conformation of actin and the rate of ATP hydrolysis, therefore regulating actin dynamics. Finally, the rate of nucleotide exchange on G-actin is relatively slow, which allows the critical concentration to increase with the number of filaments in ATP, a property largely used by the cell via the action of severing proteins.     

The plasma membrane Ca pump catalyzes the hydrolysis of ATP at low rate in the absence of Ca

The plasma membrane Ca²⁺ ATPase catalyzed the hydrolysis of ATP in the presence of millimolar concentrations of EGTA and no added Ca²⁺ at a rate near 1.5% of that attained at saturating concentrations of Ca²⁺. Like the Ca-dependent ATPase, the Ca-independent activity was lower when the enzyme was autoinhibited, and increased when the enzyme was activated by acidic lipids or partial proteolysis. The ATP concentration dependence of the Ca²⁺-independent ATPase was consistent with ATP binding to the low affinity modulatory site. In this condition a small amount of hydroxylamine-sensitive phosphoenzyme was formed and rapidly decayed when chased with cold ATP. We propose that the Ca²⁺-independent ATP hydrolysis reflects the well known phosphatase activity which is maximal in the absence of Ca²⁺ and is catalyzed by E₂-like forms of the enzyme. In agreement with this idea pNPP, a classic phosphatase substrate was a very effective inhibitor of the ATP hydrolysis.     

Intermediate complex of ATP hydrolysis and synthesis by muscle proteins

Myosin catalyzed exchange between ³²P_i and ATP in reaction medium during its enzymatic hydrolysis of ATP only by a very small amount. Addition of actin increased to a great extent the rate of incorporation of ³²P_i in the presence of Mg. Glycerinated smooth muscle fibers also exhibited the ability to exchange ³²P_i and ATP upon the application of external force (repeated stretching and releasing). A schematic mechanism of the action of actin and external force on acceleration of ³²P_i incorporation is proposed and the importance of the M*-ADP complex for force generation is suggested.     

Influence of the redox state and the activation of the chloroplast ATP synthase on proton-transport-coupled ATP synthesis/hydrolysis

The membrane-bound ATP synthase from chloroplasts can occur in different redox and activation states. In the absence of reductants the enzyme usually is oxidized and inactive, E^ox_i. Illumination in the presence of dithiothreitol leads to an active, reduced enzyme, E^red_a. If this form is stored in the dark in the presence of dithiothreitol an inactive, reduced enzyme E^red_i is formed. The rates of ATP synthesis and ATP hydrolysis catalyzed by the different enzyme species are measured as a function of ΔpH (Δψ = 0 mV). The ΔpH was generated with an acid-base transition using a rapid-mixing quenched flow apparatus. The following results were obtained. (1) The oxidized ATP synthase catalyzes high rates of ATP synthesis, v^ox_max = 400 ATP per CF₀F₁ per s. The half-maximal rate is obtained at ΔpH = 3.4. (2) The active, reduced ATP synthase catalyzes high rates of ATP synthesis, v^red_max = 400 ATP per CF₀F₁ per s. The half-maximal rate is obtained at ΔpH = 2.7. It catalyzes also high rates of ATP hydrolysis v^red_max = −90 ATP per CF₀F per s at ΔpH = 0. (3) The inactive species (both oxidized and reduced) catalyze neither ATP synthesis nor ATP hydrolysis. The activation/inactivation of the reduced enzyme is completely reversible. (4) The activation of the reduced, inactive enzyme is measured as a function of ΔpH by measuring the rate of ATP hydrolysis catalyzed by the active species. Half-maximal activation is observed at ΔpH = 2.2. (5) On the basis of these results a reaction scheme is proposed relating the redox reaction, the activation and the catalytic reaction of the chloroplast ATP synthase.     

Differential inhibition of Pi-ATP exchange in relation to ATP synthesis and hydrolysis by modification of chloroplast thylakoid membranes with glutaraldehyde

Limited modification of thylakoid membranes with glutaraldehyde inhibits the P_i-ATP exchange reaction much more than ATP synthesis or hydrolysis. More extensive modification of the membranes results in the inhibition of all activities of the ATP synthetase, but does not affect electron transport. Limited modification also does not have much effect on the tight binding of [³H]ADP or the ΔpH supported by ATP hydrolysis. The modification affects the catalytic process itself and not the activation of the latent enzyme. Cross-linking between thylakoid polypeptides is observed only after extensive treatment with glutaraldehyde, while limited modification does not result in cross-linking between polypeptides. The differential inhibition of the P_i-ATP exchange relative to ATP hydrolysis can be explained by the decrease in only one of the kinetic rate constants involved in these reactions. However, the relative insensitivity of photophosphorylation to the modification suggests that different enzyme conformations may participate in phosphorylation (light) and ATP hydrolysis or P_i-ATP exchange (dark).     

Studies on ATP hydrolysis in medium for histochemical demonstration of ATPase activity

Summary Nonenzymatic ATP hydrolysis in medium of Wachstein and Meisel for histochemical demonstration of ATPase activity was investigated. In this medium considerable amounts of phosphorus are released without the participation of the enzyme. ATP hydrolysis in Wachstein-Meisel's medium increase with the concentration of Pb⁺⁺ and decrease at its small concentrations. The degree of ATP hydrolysis appeared to increase with increase both temperature and pH. At high concentration of ATP (5.76 mM) the degree of ATP hydrolysis in Wachstein-Meisel's medium is lower than at 1.44 mM ATP. 10.0 mM Ca⁺⁺ or 3.6 mM Fe⁺⁺ speed up ATP hydrolysis after 30- and 60-minute incubation. In the presence of 3.6 mM Co⁺⁺ or 2.6 mM Cu⁺⁺ ATP hydrolysis in Wachstein-Meisel's medium increased throughout the whole period examined. On the contrary, 3.6 mM Fe⁺⁺⁺ decreases ATP hydrolysis in this medium.10.0 mM F^– raises the degree of ATP hydrolysis which is, however, lowered in the presence of 2.5 mM pCMB or 3.6 mM KCN. 2.0 mM cysteine highly inhibits the process of nonenzymatic ATP hydrolysis in Wachstein-Meisel's medium.These data show that the histochemical reaction for ATPase activity in Wachstein-Meisel's medium does not originate exclusively from the hydrolysis of ATP in the presence of Pb⁺⁺, but take rise, above all, as a result of an enzymatic reaction.     

Stereochemistry of binding of thiophosphate analogs of ATP and ADP to carbamate kinase, glutamine synthetase, and carbamoyl-phosphate synthetase

Thiophosphate analogs of adenine nucleotides were used to establish the absolute stereochemistry of nucleotide substrates in the reactions of carbamate kinase (Streptococcus faecalis), unadenylylated glutamine synthetase (Escherichia coli), and carbamoyl-phosphate synthetase (E. coli). ³¹P NMR was used to determine that carbamate kinase uses the B isomer of Ado-5′-(2-thioPPP) in the presence of Mg²⁺. The stereospecificity of the reaction with carbamate kinase was not reversed by Cd²⁺ suggesting that the metal ion does not bind to the β-phosphoryl group or that both Mg²⁺ and Cd²⁺ bind to the sulfur atom. Carbamate kinase uses both A and B isomers of Ado-5′-(1-thioPP) with Mg²⁺ and Cd²⁺. We have previously reported that carbamoyl-phosphate synthetase uses the A isomer of Ado-5′-(2-thioPPP) at both ATP sites with Mg²⁺ (Raushel et al., 1978J. Biol. Chem.253, 6627). Current experiments show that the stereospecificity is reversed by Cd^2? and that both A and B isomers are used when Zn²⁺ is present. With Ado-5′-(1-thioPPP), the B isomer is used with Mg²⁺, the A isomer with Cd²⁺, and both isomers with Zn²⁺. Neither carbamate kinase nor carbamoyl-phosphate synthetase utilized Co(III)(NH₃)₄ATP as a substrate and thus we can only speculate that the Δ chelate ring configuration is the chelate structure utilized by carbamoyl-phosphate synthetase (based on the analogy between thiophosphate-ATP analogs and Co³⁺-ATP analogs utilized by hexokinase (E. K. Jaffe, and M. Cohn, 1978Biochemistry17, 652). If the sulfur of the β-phosphoryl of Ado-5′-(2-thioPPP) binds to the metal ion with carbamate kinase, then the Δ chelate ring is also used in this enzyme that catalyzes one of the steps in the overall reaction catalyzed by carbamoyl-phosphate synthetase. Glutamine synthetase reacts with the B isomer of both Ado-5′-(2-thioPPP) and Ado-5′-(1-thioPPP) in the presence of Mg²⁺. When Co²⁺ is used with this enzyme the A and B isomers of both thio-ATP compounds are substrates. Co(III)(NH₃)₄ATP is not a substrate for glutamine synthetase. Glutamine synthetase is therefore different from the two previously mentioned enzymes in that it used the opposite A ring configuration for the metal-ATP chelate.     

Characterization of an ATP diphosphohydrolase activity (APYRASE,EC 3.6.1.5) in rat blood platelets

In the present report we describe an apyrase (ATP diphosphohydrolase, EC 3.6.1.5) in rat blood platelets. The enzyme hydrolyses almost identically quite different nucleoside di- and triphosphates. The calcium dependence and pH requirement were the same for the hydrolysis of ATP and ADP and the apparent Km values were similar for both Ca²⁺-ATP and Ca²⁺-ADP as substrates. Ca²⁺-ATP and Ca²⁺-ADP hydrolysis could not be attributed to the combined action of different enzymes because adenylate kinase, inorganic pyrophosphatase and nonspecific phosphatases were not detected under our assay conditions. The Ca²⁺-ATPase and Ca²⁺-ADPase activity was insensitive to ATPase, adenylate kinase and alkaline phosphatase classical inhibitors, thus excluding these enzymes as contaminants. The results demonstrate that rat blood platelets contain an ATP diphosphohydrolase involved in the hydrolysis of ATP and ADP which are vasoactive and platelet active adenine nucleotides.     

ATP hydrolysis and synthesis by the membrane-bound ATP synthetase complex of Methanobacterium thermoautotrophicum.

The membrane-bound ATP synthetase complex of Methanobacterium thermoautotrophicum showed maximum activity for ATP hydrolysis at pH 8, at temperatures between 65 and 70 degrees C, and at an ATP-Mg2+ ratio of 0.5. Anaerobic conditions were not prerequisite for enzyme activity. The enzyme showed a Km value for ATP of 2 mM, and activity was Mg2+ dependent; Mn2+, Co2+, Ca2+, and Zn2+ could replace Mg2+ to some extent. Other nucleoside triphosphates could be hydrolyzed. N,N'-dicyclohexylcarbodiimide inhibited ATP hydrolysis. A proton-motive force, artificially imposed by a pH shift or valinomycin, resulted in ATP synthesis in whole cells. The ATP synthetase complex of the thermophilic methanogenic bacterium is similar to those described in aerobic and anaerobic microorganisms.     

Coupling of proteolysis to ATP hydrolysis uponEscherichia coli lon protease functioning: I. kinetic aspects of ATP hydrolysis

Some aspects of theEscherichia coli Lon protease ATPase function were studied around the optimum pH value. It was revealed that in the absence of the protein substrate the maximum ATPase activity of the enzyme is observed at an equimolar ratio of ATP and Mg²⁺ ions in the area of their millimolar concentrations. Free components of the substrate complex (ATP-Mg)²⁻ inhibit the enzyme ATPase activity. It is hypothesized that the effector activity of free Mg²⁺ ions is caused by the formation of the “ADP-Mg-form” of ATPase centers. It was shown that the activation of ATP hydrolysis in the presence of the protein substrate is accompanied by an increase in the affinity of the (ATP-Mg)²⁻ complex to the enzyme, by an elimination of the inhibiting action of free Mg²⁺ ions without altering the efficiency of catalysis of ATP hydrolysis (based on thek _cat value), and by a change in the type of inhibition of ATP hydrolysis by the (ADP-Mg)⁻ complex (without changing theK _i value). Interaction of the Lon protease protein substrate with the enzyme area located outside the peptide hydrolase center was demonstrated by a direct experiment.     

Enhancement of the rate of pyrophosphate hydrolysis by nonenzymatic catalysts and by inorganic pyrophosphatase

To estimate the proficiency of inorganic pyrophosphatase as a catalyst, ³¹P NMR was used to determine rate constants and thermodynamics of activation for the spontaneous hydrolysis of inorganic pyrophosphate (PP_i) in the presence and absence of Mg²⁺ at elevated temperatures. These values were compared with rate constants and activation parameters determined for the reaction catalyzed by Escherichia coli inorganic pyrophosphatase using isothermal titration calorimetry. At 25 °C and pH 8.5, the hydrolysis of MgPP_i²⁻ proceeds with a rate constant of 2.8 × 10⁻¹⁰ s⁻¹, whereas E. coli pyrophosphatase was found to have a turnover number of 570 s⁻¹ under the same conditions. The resulting rate enhancement (2 × 10¹²-fold) is achieved entirely by reducing the enthalpy of activation (ΔΔH^‡ = −16.6 kcal/mol). The presence of Mg²⁺ ions or the transfer of the substrate from bulk water to dimethyl sulfoxide was found to increase the rate of pyrophosphate hydrolysis by as much as ∼10⁶-fold. Transfer to dimethyl sulfoxide accelerated PP_i hydrolysis by reducing the enthalpy of activation. Mg²⁺ increased the rate of PP_i hydrolysis by both increasing the entropy of activation and reducing the enthalpy of activation.     

The mechanism of ATP–G-actin hydrolysis in Mg<Superscript>2+</Superscript>-containing solutions

NMR proton spectra were recorded in the range of proton resonance in the nucleotide aromatic ring of monomeric ATP–G-actin and the Mg²⁺–ATP–G-actin solutions in D2O to study the mechanism of ATP–G-actin hydrolysis and its role in F-actin formation in Mg²⁺-containing solutions. The experimental data show variations in the proton chemical shifts of the H2 and H8 peaks and splitting of the H8 resonance peak of G-actin-bound ATP adenine caused by interaction with magnesium dication. The observed variations in spectra are explained by hydrolysis of monomeric ATP–G-actin to ADP–G-actin, which is regarded as the initial stage of the G-actin to F-actin transformation.     

The standard Gibbs free energy change of hydrolysis of alpha-D-ribose 1-phosphate

The standard Gibbs free energy change of hydrolysis of α-d-ribose 1-phosphate has been measured at pH 7.0, ionic strength 0.1 m, and 25 °C by combining the corresponding values of the two following reactions: adenosine + H₂O ág adenine + ribose (ΔG^0′ = ?2.3 ± 0.1 kcal/mol), catalyzed by adenosine nucleosidase, and ribose 1-phosphate + adenine ág adenosine + P_i (ΔG^0′ = ?3.1 ± 0.1 kcal/mol), catalyzed by adenosine phosphorylase. The standard Gibbs free energy changes were calculated for both reactions from the equilibrium constant. A value of -5.4 ± 0.15 kcal/mol, comparable to that of other hemiacetal phosphoric esters, was obtained for the hydrolysis of ribose 1-phosphate.     

Adenosine triphosphate (ATP) hydrolysis promoted by cobalt(III).Participation of polynuclear metal complexes

Complex formation between ATP (adenosine 5′-triphosphate) and tn₂CO^III(aq) (tn = trimethylenediamine) and resulting hydrolysis of the ATP to ADP (adenosine 5′-diphosphate), AMP (adenosine 5′-monophosphate), PP_i (pyrophosphate), and P_i (orthophosphate) have been examined by means of ³¹P nmr. With ATP ～0.1 M and tn₂Co^III(aq) up to 0.3 M, complex formation was promoted by equilibrating solutions for a period at pH 4, after which hydrolysis was allowed to proceed at each of several pHs in the range 5 to 9 prior to quenching by addition of strong base. With ATP 0.01 M and tn₂Co^III(aq) up to 0.08 M, the above procedure was followed in some cases; in other experiments the pH of each ATP/tn₂Co^III(aq) solution was adjusted immediately to a value in the range 5 to 9 with the remainder of the procedure as before. In most cases the hydrolysis was at 25°C, but temperature dependence was also examined. The integrals for the β-phosphorus resonance have been used to analyze for ATP in the quenched solutions; independent measurements of ATP by an enzyme/spectrophotometric method (Bergmeyer) gave similar results. Cobalt to ATP molar ratios up to 1 produce tn₂Co^IIIATP as the predominant ATP complex; this 1:1 complex shows no detectable acceleration in hydrolysis compared to free ATP. Cobalt to ATP molar ratios of ?1 lead to complexes of type (tn₂Co^III)₂ATP and (tn₂Co^III)₃ATP, which exhibit greatly enhanced reactivity towards ATP hydrolysis. At a 2:1 molar ratio (0.1 or 0.01 M ATP), the enhancement is rate is ～10⁵ at pH 7 where the rate is a maximum (comparison for 25°C); at higher molar ratios the rate enhancements are even greater. The results support the view that effective metal ion catalysis of ATP hydrolysis requires formation of reactive species involving more than one metal ion per ATP.     

Direct fluorimetric measurement on the hydrolysis of β-naphthyl triphosphate, a fluorescent ATP analog, by heavy meromyosin

A fluorescent ATP analog, β-naphthyl triphosphate, was hydrolyzed to β-naphthyl diphosphate and orthophosphate by heavy meromyosin ATPase. In the process of hydrolysis the fluorescence intensity of β-naphthyl triphosphate changed remarkably. Thus, the rate of β-naphthyl triphosphate hydrolysis is evaluated directly and continuously by measuring the time course of fluorescence intensity.In the presence of Ca²⁺, the Michaelis constant (K_m) of β-naphthyl triphosphate hydrolysis by heavy meromyosin was similar to that of ATP hydrolysis. While, in the presence of Mg²⁺ the K_m of β-napthyl triphosphate hydrolysis was 9.0·10⁻⁶ M, much larger than the value of ATP hydrolysis, indicating that the apparent affinity of the enzyme for β-naphthyl triphosphate is less than that for ATP.The pH dependence of β-naphthyl triphosphatase activity resembled that of ATPase activity, suggesting a similarity in the mechanism of hydrolysis of the two substrates.     

Opening of mitochondrial K+ channels increases ischemic ATP levels by preventing hydrolysis

Mitochondrial ATP-sensitive K⁺ channels (mitoK_ATP) have been proposed to mediate protection against ischemic injury by increasing high-energy intermediate levels. This study was designed to verify if mitochondria are an important factor in the loss of cardiac ATP associated to ischemia, and determine the possible role of mitoK_ATP in the control of ischemic ATP loss. Langendorff-perfused rat hearts subjected to ischemia were found to have significantly higher ATP contents when pretreated with oligomycin or atractyloside, indicating that mitochondrial ATP hydrolysis contributes toward ischemic ATP depletion. MitoK_ATP opening induced by diazoxide promoted a similar protection against ATP loss. Diazoxide also inhibited ATP hydrolysis in isolated, nonrespiring mitochondria, an effect accompanied by a drop in the membrane potential and Ca²⁺ uptake. In hearts subjected to ischemia followed by reperfusion, myocardial injury was prevented by diazoxide, but not atractyloside or oligomycin, which, unlike diazoxide, decreased reperfusion ATP levels. Our results suggest that mitoK_ATP-mediated protection occurs due to selective inhibition of mitochondrial ATP hydrolysis during ischemia, without affecting ATP synthesis after reperfusion.     

Ca-stimulated, Mg-independent ATP hydrolysis and the high affinity Ca-pumping ATPase. Two different activities in rat kidney basolateral membranes

The Mg²⁺-dependency of Ca²⁺-induced ATP hydrolysis is studied in basolateral plasma membrane vesicles from rat kidney cortex in the presence of CDTA and EGTA as Mg²⁺- and Ca²⁺-buffering ligands. ATP hydrolysis is strongly stimulated by Mg²⁺ with a K_m of 13 μ M in the absence or presence of 1 μ M free Ca²⁺. At free Mg²⁺ concentrations of 1 μ M and lower, ATP hydrolysis is Mg²⁺ -independent, but is strongly stimulated by submicromolar Ca²⁺ concentrations K_m = 0.25 μM, V_max = 24 μmol P_i/h per mg protein). The Ca²⁺-stimulated ATP hydrolysis strongly decreases at higher Mg²⁺ concentrations. The Ca²⁺-stimulated Mg²⁺-independent ATP hydrolysis is not affected by calmodulin or trifluoperazine and shows no specificity for ATP over ADP, ITP and GTP. In contrast, at high Mg²⁺ concentrations calmodulin and trifluoperazine affect the high affinity Ca²⁺-ATPase activity significantly and ATP is the preferred substrate. Control studies on ATP-dependent Ca²⁺-pumping in renal basolaterals and on Ca²⁺-ATPase in erythrocyte ghosts suggest that the Ca²⁺-pumping enzyme requires Mg²⁺. In contrast, a role of the Ca²⁺-stimulated Mg²⁺-independent ATP hydrolysis in active Ca²⁺ transport across basolateral membranes is rather unlikely.     

A structural perspective on the PP-loop ATP pyrophosphatase family

Derived from an ancient ATP-hydrolyzing Rossmann-like fold protein, members of the PP-loop ATP pyrophosphatase family feature an absolutely conserved P-loop-like “SxGxDS/T” motif used for binding and presenting ATP for substrate adenylylation (AMPylation). Since the first family member was reported more than 20 years ago, numerous representatives catalyzing very diverse reactions have been characterized both functionally and structurally. The availability of more than 100 high quality structures in the protein data bank provides an excellent opportunity to gain structural insights into the generally conserved catalytic mechanism and the uniqueness of the reactions catalyzed by family members. In this work, we conducted a thorough database search for the PP-loop ATP pyrophosphatase family members, resulting in the most comprehensive and up-to-date collection that includes 18 enzyme families. Structure comparison of representative family members allowed us to identify common structure features in the core catalytic domain, as well as four highly variable regions that define the unique chemistry for each enzyme family. The newly identified enzymes, particularly those from pathogens, warrant further research to enlarge the scope of this ever-expanding and highly diverse enzyme superfamily for use in potential bioengineering and biomedical applications.     

The use of some ATP analogues for the analysis of the mechanism of ATP hydrolysis by the organic pyrophosphatase of wheat seedlings.

The present experiments have shown that the activity of organic pyrophosphatase from microsomes of wheat seedlings is strongly inhibited by alpha, beta-methylene and gamma-thio-ATP derivatives, which inhibit the hydrolysis of both ATP and ADP. It has been found that the main products of ATP hydrolysis are not ADP but AMP and orthophosphate. The rate of hydrolysis of ATP is not increased by addition of ADP to the incubation medium. The ratio of the reaction products, Pi and AMP, amounts to 1.7-1.8 with ATP as substrate and is close to 1.0 with ADP.