Enzyme-bound ADP Research Articles

Activation of mitochondrial ATP-dependent protease by peptides and proteins.

We examined the effect of peptides or protein on the proteolytic and ATPase activities of mitochondrial ATP-dependent LON protease purified from bovine adrenal cortex. Peptides/proteins including angiotensin I which stimulated ATPase activity without hydrolysis of any peptide bonds stimulated proteolysis of 125I-labeled substrates at low concentrations; whereas at high concentrations they competitively inhibited proteolysis, thus displaying a biphasic activity profile. All peptides and proteins thus examined stimulated degradation of 125I-labeled substrates. When an ATP analog was substituted for ATP, only inhibition; i.e., no stimulation, of proteolysis by unlabeled peptides was observed. Without activator peptides, degradation of [125I] peptides was higher in the presence of an ATP analog than that in the presence of ATP. ADP, a product of the ATPase reaction, inhibited the proteolytic activity in the absence of an activator peptide but not in its presence. From analogy to E. coli ATP-dependent protease La (LON), we suggest that the activator peptides stimulated the proteolysis by releasing enzyme-bound ADP.

Perspectives on the Mechanism of ATP Hydrolysis by Nitrogenase

The chemical mechanism of ATP hydrolysis by nitrogenase is discussed in terms of results from isotopic exchange studies using [18O4]-Pi, b,g-[18O]-ATP, and g-[18O4]-ATP as probes of reversibility (ATP = ADP + Pi) and of the freedom of rotation around the O-Pb bond in enzyme-bound ADP.

3′-Phosphorylated Nucleotides Are Tight Binding Inhibitors of Nucleoside Diphosphate Kinase Activity

Nucleoside diphosphate (NDP) kinase catalyzes the phosphorylation of ribo- and deoxyribonucleosides diphosphates into triphosphates. NDP kinase is also involved in malignant tumors and was shown to activate in vitro transcription of the c-myc oncogene by binding to its NHE sequence. The structure of the complex of NDP kinase with bound ADP shows that the nucleotide adopts a different conformation from that observed in other phosphokinases with an internal H bond between the 3'-OH and the beta-O made free by the phosphate transfer. We use intrinsic protein fluorescence to investigate the inhibitory and binding potential of nucleotide analogues phosphorylated in 3'-OH position of the ribose to both wild type and F64W mutant NDP kinase from Dictyostelium discoideum. Due to their 3'-phosphate, 5'-phosphoadenosine 3'-phosphate (PAP) and adenosine 3'-phosphate 5'-phosphosulfate (PAPS) can be regarded as structural analogues of enzyme-bound ADP. The KD of PAPS (10 microM) is three times lower than the KD of ADP. PAPS also acts as a competitive inhibitor toward natural substrates during catalysis, with a KI in agreement with binding data. The crystal structure of the binary complex between Dictyostelium NDP kinase and PAPS was solved at 2.8-A resolution. It shows a new mode of nucleotide binding at the active site with the 3'-phosphate of PAPS located near the catalytic histidine, at the same position as the gamma-phosphate in the transition state. The sulfate group is directed toward the protein surface. PAPS will be useful for the design of high affinity drugs targeted to NDP kinases.

Synthesis and release of ATP by soluble mitochondrial F1 in complex with its inhibitor protein during dimethylsulfoxide-water transitions.

Soluble mitochondrial F1 and F1 in complex with the natural ATPase inhibitor protein (F1-IP) catalyze the spontaneous synthesis of [gamma-32P]ATP from medium [32P]phosphate and enzyme-bound ADP when incubated in media with dimethylsulfoxide (Me2SO); under these conditions, the synthesized [gamma-32P]ATP is not released into the media, it remains tightly bound to the enzymes [Gómez-Puyou, A., Tuena de Gómez-Puyou, M. & de Meis, L. (1986) Eur. J. Biochem. 159, 133-140]. Some of the characteristics of the synthesized [gamma-32P]ATP were studied in F1 and F1-IP (ATPase activities of 70 and 1-3 micromol x min(-1) x mg(-1), respectively). In Me2SO media, gamma-phosphate of synthesized ATP in F1 or F1-IP exchanges with medium phosphate. From the rates of the exchange reaction, the half-times for hydrolysis of the synthesized ATP in F1 and F1-IP were calculated: 45 min and 58 min for F1 and F1-IP, respectively. The course that synthesized [gamma-32P]ATP follows after dilution of the Me2SO synthetic mixture with aqueous buffer was determined. After dilution, the half-life of synthesized ATP in F1 was less than 1 min. In F1-IP, ATP was also hydrolyzed, but at significantly lower rates. In F1-IP, dilution also produced release of the synthesized [gamma-32P]ATP. This was assayed by the accessibility of [gamma-32P]ATP to hexokinase. About 25% of [gamma-32P]ATP synthesized in F1-IP, but not in F1, was released into the media after dilution with aqueous buffer that contained 20 mM phosphate. Release of tightly bound ATP required the binding energy of phosphate and solvation of F1-IP, however, the particular kinetics of F1-IP were also central for medium ATP synthesis in the absence of electrochemical H+ gradients.

Mechanism of carbamoyl phosphate synthetase from Escherichia coli--binding of the ATP molecules used in the reaction and sequestration by the enzyme of the ATP molecule that yields carbamoyl phosphate.

The conflicting data on the binding of the two molecules of ATP that are involved in the overall reaction catalyzed by carbamoyl-phosphate synthetase (CPS) of Escherichia coli, and a mechanism recently proposed for this reaction, has led us to reexamine ATP binding using pulse/chase techniques. With [gamma-32P]ATP and bicarbonate in the pulse solution, there is a positive intercept at zero time of approximately 1 mol Pi/mol CPS in the plot of 32Pi formation against time, irrespective of whether the incubation is terminated by the addition of acid or by addition of a chase solution containing glutamine, excess unlabeled ATP and bicarbonate. The intercept is decreased to about 50% if the excess unlabeled ATP is added prior to the addition of the glutamine. These are the expected results if the intercept reflects the reversible formation of enzyme-bound ADP and carboxyphosphate. Approximately 0.6 mol carbamoyl [32P]phosphate/mol enzyme is formed in these experiments when the pulse step is terminated by addition to the chase solution. The ATP molecule that provides the phosphoryl group of carbamoyl phosphate, therefore, also binds to the enzyme in the absence of ammonia or glutamine and reacts in the chase to give carbamoyl phosphate before it can dissociate from the enzyme. At 1 mM ATP, the binding of both ATP molecules is essentially complete at 2.5 s, but the dissociation of the ATP that yields carbamoyl phosphate is extremely slow (t(1/2) of about 6 min at 22 degrees C; HCO3-, 40 mM), although it is faster in the absence of bicarbonate. The extreme sequestration from the aqueous environment of this ATP allows the enzyme-ATP complex to be separated from the surrounding ATP by centrifugal gel filtration. After two successive steps of gel filtration through Sephadex G-50 equilibrated with unlabeled ATP and bicarbonate, the majority of the radioactivity remaining in the solution is bound to the enzyme and is released as [gamma-32P]ATP if acid is added, or is converted to carbamoyl [32P]phosphate by addition to chase solution, without concomitant release of 32Pi. K+ is necessary in the pulse solution, but not in the chase solution, to demonstrate this binding. These findings and other confirmatory experiments demonstrate conclusively that, in the presence of K+, both ATP molecules bind to the enzyme in the absence of ammonia or glutamine. The bound ATP that yields Pi in the overall reaction is replaced relatively rapidly by exchange and by hydrolysis in the bicarbonate-dependent ATPase activity of the enzyme, whereas the bound ATP that provides the phosphoryl group of carbamoyl phosphate is replaced very slowly. The temporal pattern of carbamoyl [32P]phosphate formation from [gamma-32P]ATP, in pulse/chase experiments in which a small concentration of ammonia is added to the pulse solution, shows that, in the normal enzyme reaction, this last ATP molecule binds to the enzyme before ammonia. These findings exclude a recently proposed mechanism [Kothe, M., Eroglu, B., Mazza, H., Samudera, H. & Powers-Lee, S. (1997) Proc. Natl Acad. Sci. USA 94, 12348-12353] in which a single molecule of ATP bound at the catalytic center phosphorylates bicarbonate and provides the phosphoryl group of carbamoyl phosphate. A mechanism in which a single ATP molecule binds, followed by the binding of bicarbonate and ammonia (from glutamine) and the release of Pi before the second molecule of ATP is bound is also excluded. We have previously reported very similar findings for carbamoyl-phosphate synthetase (ammonia), strongly suggesting that the different types of CPS share a common mechanism. The virtual sequestration of the ATP that provides the phosphoryl group of carbamoyl phosphate is consistent with a palmate-binding site, with the nucleotide bound within a beta-sheet sandwich, and a loop closure mechanism triggered by the binding of bicarbonate or the formation of carboxyphosphate.

Isotope exchange studies on the Escherichia coli selenophosphate synthetase mechanism.

Selenophosphate synthetase, the Escherichia coli selD gene product, is a 37-kDa protein that catalyzes the synthesis of selenophosphate from ATP and selenide. In the absence of selenide, ATP is converted quantitatively to AMP and two orthophosphates in a very slow partial reaction. A monophosphorylated enzyme derivative containing the gamma-phosphoryl group of ATP has been implicated as an intermediate from the results of positional isotope exchange studies. Conservation of the phosphate bond energy in the final selenophosphate product is indicated by its ability to phosphorylate alcohols and amines to form O-phosphoryl- and N-phosphoryl-derivatives. To further probe the mechanism of action of selenophosphate synthetase, isotope exchange studies with [8-14C]ADP or [8-14C]AMP and unlabeled ATP were carried out, and 31P NMR analysis of reaction mixtures enriched in H218O was performed. A slow enzyme-catalyzed exchange of ADP with ATP observed in the absence of selenide implies the existence of a phosphorylated enzyme and further supports an intermediary role of ADP in the reaction. Under these conditions ADP is slowly converted to AMP. Incorporation of 18O from H218O exclusively into orthophosphate in the overall selenide-dependent reaction indicates that the beta-phosphoryl group of the enzyme-bound ADP is attacked by water with liberation of orthophosphate and formation of AMP. Based on these results and the failure of the enzyme to catalyze an exchange of labeled AMP with ATP, the existence of a pyrophosphorylated enzyme intermediate that was postulated earlier can be excluded.

SYNTHESIS OF A SELF‐CONTAINED CONCEPT OF THE MOLECULAR MECHANISM OF ENERGY INTERCONVERSION BY H+‐TRANSPORTING ATP SYNTHASE

The original aim of the review has been to probe into the validity of the paradigm on the high energy-carrier function of ATP. It seemed to be called into question on the basis of findings with H(+)-transporting ATP synthase suggesting the formation of ATP from ADP and Pi without energy input. Thus, ATP appeared as a low-energy compound. Starting from the current, rich knowledge of the molecular structure and the inviting thinking on the mechanism of H(+)-transporting ATP synthase, we have endeavoured to freshly interpret and integrate the pertinent observations in the light of the comprehensively derived model of the molecular mechanism of energy interconversion by Na+/K(+)-transporting ATPase. In this way, we have uncovered the common mechanistic elements of the two energy-interconverting enzymes. The emerging purpose of the present paper has been the 'synthesis' of a self-contained concept of the molecular mechanism of the interconversion of electrochemical and chemical Gibbs energies by H(+)-transporting ATP synthase. The outcome is reflected in the following tentative evaluations. 1. In ATP hydrolysis, the great Gibbs energy change which is observed in solution, is largely conserved by the F1 sector of ATP synthase as mechanical Gibbs energy in the enzyme's protein fabric, so that it can be utilized in the resynthesis of ATP from enzyme-bound ADP and Pi. The plainly measured low Gibbs energy change results from large compensating enthalpy and entropy changes that reflect the underlying changes in protein conformation. 2. In stoichiometric ATP synthesis by F1 sector from ADP and Pi bound to the catalytic centre, their intrinsic binding energy brings about a loss of peptide chain entropy that makes possible an entropy-driven ATP formation. 3. The driving force for ATP synthesis cannot be the high Gibbs energy change on binding of product ATP; the tight ATP-enzyme complex rather is a low Gibbs energy intermediate from which escape is difficult. 4. The catalytic centre exists either in an open state unable to firmly bind the substrate-product couple, or in a closed state protecting formed ATP from facile hydrolysis by ambient water. 5. The cleft closure, induced by binding of Pi and ADP or ATP, does not necessarily need external energy supply, because the cleft closure proceeds from rigid domain rotations which can occur rather spontaneously. In further analogy to adenylate kinase, the driving force of this domain movement presumably comes from the electrostatic interactions between phosphate moieties and arginine side chains in the catalytic centre.(ABSTRACT TRUNCATED AT 400 WORDS)

Transport protons do not participate in ATP synthesis/hydrolysis at the nucleotide binding site of the H +-ATPase from chloroplasts

The H +-ATPase from chloroplasts, CF 0P 1, was brought into the active, reduced state by illumination of thylakoids in the presence of thioredoxin and dithiothreitol. Uni-site ATP synthesis was initiated by the addition of 20 nM [α- 32P]ADP, and enzyme-bound and free nucleotides were separated by a pressure column. The ratio of enzyme-bound ADP to ATP was 0.55 ± 0.05. In a second experiment, uni-site ATP hydrolysis under energized conditions was initiated by the addition of 36 nM [α- 32P]ATP; enzyme-bound and free nucleotides were separated by a pressure column. Both procedures were carried out under continuous illumination. The ratio of enzyme-bound ADP to ATP was 0.46 ± 0.04. In a third experiment, uni-site ATP hydrolysis under de-energized conditions was initiated by the addition of 39 nM [α- 32P]ATP and NH 4Cl/valinomycin in the absence of illumination. Free and enzyme-bound nucleotides were separated also by a pressure column. The ratio of enzyme-bound ADP to ATP was 0.43 ± 0.02. This ratio was always the same irrespective of whether the reaction runs in the synthesis or the hydrolysis direction. Furthermore, the ratio does not depend on the membrane energization. We conclude, therefore, that the protons are not directly involved in the reaction at the catalytic site.

The synthesis of enzyme-bound ATP by the F 1-ATPase from the thermophilic bacterium PS3 in the presence of organic solvents

Synthesis of enzyme-bound ATP was demonstrated with purified TF 1 (F 1-ATPase from thermophilic bacterium PS3) from medium inorganic phosphate (P i) and enzyme-bound ADP in the presence of organic solvents such as dioxane, ethanol, dimethylformamide, methanol, acetone, acetonitrile or ethyleneglycol. The optimal concentrations of dimethylformamide, ethanol or methanol were 50%, 30% and 40% and the half-maximal concentrations of P i were 13 mM, 20 mM and 18 mM, respectively. Thus it is evident that the effect of dimethylsulfoxide on TF 1 to form enzyme-bound ATP [8] is not due to a specific interaction between dimethylsulfoxide and the enzyme, but to a decrease in polarity of the medium. In the presence of methanol, the dependence of ATP synthesis on various divalent metal ions was compared to that for the ATP-hydrolyzing activity and the ATP-driven proton-translocating activity of TF 1. While Mn 2+, Co 2+, Zn 2+ and Cd 2+ are as effective as Mg 2+ for the ATP-hydrolyzing activity of TF 1, Zn 2+ and Cd 2+ are either less or not effective for proton translocation and for ATP synthesis. This result appears to be consistent with the idea that the TF 1-ATP complex formed in organic solvents represents one of the intermediates in the reaction sequences of ATP synthesis by H +-ATPase using the proton gradient.

The synthesis of enzyme-bound ATP by the F1-ATPase from the thermophilic bacterium PS3 in 50% dimethylsulfoxide

Purified TF1 (F1-ATPase from a thermophilic bacterium PS3) synthesizes enzyme-bound ATP from medium Pi and enzyme-bound ADP in the presence of 50% dimethylsulfoxide (DMSO). Once ATP was formed on the enzyme, it was not released even after removal of DMSO and Pi from the solution. The half maximal concentration of medium Pi for ATP synthesis was 1mM. The pH optimum for enzyme-bound ATP formation was about 6.5. Under the optimum conditions, a yield of up to 0.8 mol of ATP/mol of TF1 was obtained.

On the enzymic mechanism of oxidative phosphorylation.

Oxidative phosphorylation, like substrate-level phosphorylation, involves oxidative conversion of inorganic phosphate to a reactive species followed by interaction of this species with enzyme-bound ADP to form enzyme-bound ATP. The reactive species in a phosphoryl ester in substrate-level phosphorylation and phosphonium ion of orthophosphate in oxidative phosphorylation. The coupled synthesis is mediated by a combination of two classical enzymes in substrate-level phosphorylation and by a set of energy-coupled enzymes in oxidative phosphorylation. The full range of experimental evidence supporting this proposed enzymic mechanism of oxidative phosphorylation is presented as well as the rationalization of phenomena that hitherto have eluded explanation.

Mechanism of activation of bicarbonate ion by mitochondrial carbamoyl-phosphate synthetase: formation of enzyme-bound adenosine diphosphate from the adenosine triphosphate that yields inorganic phosphate.

The mechanism of the reaction catalyzed by rat liver mitochondrial carbamoyl-phosphate synthetase has been studied by using [beta-18O2]ATP and HC18O-3, monitoring the isotopic composition of adenosine triphosphate (ATP) and inorganic phosphate (Pi) by high-resolution 31P NMR spectroscopy. In the presence of both HCO3- and acetylglutamate, the enzyme catalyzes the exchange of oxygen atoms between the beta, gamma bridging and the beta nonbridging positions of ATP. Addition of NH3 stops the exchange, Pi released by the ATPase activity of the enzyme in the absence of NH3 contains one oxygen atom from HC18O3- but there is no incorporation of 18O into ATP. There is no significant incorporation of [14C]ADP or 32Pi into ATP. It is concluded that in the enzyme-ATPA.HCO30.ATPB complex formed in the presence of ATP and HCO3- there is reversible transfer of the gamma-PO3 group of ATPA (the molecule that yields Pi) to HCO3- without dissociation of products. The beta-PO3 of the enzyme-bound ADP that is formed can rotate. Virtually all of the complex appears to be in the form in which ATPA is cleaved, but in the absence of NH3, ATP is reconstituted and dissociates from the complex on at least 75% of the occasions. On the remainder, the carbonyl phosphate is cleaved in an irreversible process that yields Pi and a low-energy form of carbonic acid (probably HCO3-). NH3 reacts rapidly and irreversibly with the complex, and at saturation the rate (greater than 10 times the rate of Pi release in the absence of NH3) is sufficient to prevent dissociation of ATPA. In the absence of HCO3- an enzyme-ATPA.ATPB complex is formed, but cleavage of the bond between beta, gamma bridging oxygen and P gamma of ATPA does not occur.

Synthesis of bound adenosine triphosphate from bound adenosine diphosphate by the purified coupling factor 1 of chloroplasts. Evidence for direct involvement of the coupling factor in this "adenylate kinase-like" reaction

Electrophoretically homogeneous coupling factor 1 from spinach chloroplasts binds ADP and converts the bound ADP to bound ATP and AMP. That this transphosphorylation of enzyme-bound ADP is catalyzed by the coupling factor itself, and not be a conventional adenylate kinase which might possibly contaminate preparations of the coupling factor, is supported by the following evidence. 1. The procedure for isolatio of the coupling factor is designed to separate this large (approximately 13 S) enzyme from the smaller (4.2 S) conventional adenylate kinase of spinach chloroplasts. The conventional adenylate kinase cannot be detected in purified preparations of the coupling factor by biochemical assay or by polyacrylamide gel electrophoresis. 2. The activity of spinach adenylate kinase is completely dependent upon magnesium ions. However, the production of bound ATP and AMP from bound ADP by the coupling factor can be assayed in the total absence of added magnesium ions or even in the presence of added EDTA. 3. Comparative studies with inhibitors show that the coupling factor can produce bound ATP from ADP under conditions where the activity of adenylate kinase is strongly inhibited. Conversely, the coupling factor is prevented from synthesizing bound ATP from ADP under other conditions where the conventional adenylate kinase has high levels of activity. 4. AMP, when added in solution to the coupling factor, does not bind to this enzyme, even in the presence of APT. Thus, it is unlikely that the appearance of AMP bound to the coupling factor after its incubation with ADP is due to the production of free AMP by contaminating adenylate kinase. These results demonstrate that the isolated, homogeneous coupling factor from spinach chloroplasts has the intrinsic capacity to perform a phosphoryl group transfer between two bound ADP molecules and thus to synthesize ATP. This reaction may have an important role in the photosynthetic production of ATP by the chloroplast, as is discussed in this communication.

A stereochemical method for detection of ATP terminal phosphate transfer in enzymatic reactions. Glutamine synthetase.

An isotope scrambling method is described for the detection of transient [Enz:ADP:P-X] formation from [18O]ATP in ATP-coupled enzyme reactions. The method makes use of torsional symmetry of the newly formed (see article) group in ADP. [18 O]ATP labeled in the betagama bridge oxygen was incubated with enzyme and reversible cleavage of the PbetaO -- Pgamma bond was detected by the appearance of 18O in the beta nonbridge oxygens of the ATP pool. Experiments with sheep brain and Escherichia coli glutamine synthetases show that cleavage of ATP of enzyme-bound ADP and P-X requires glutamate. The exchange catalyzed by the E. coli enzyme with glutamate occurs in the absence of ammonia and is partially inhibited by added NH4Cl, as expected if the exchange is in the mechanistic pathway for glutamine synthesis. The results provide kinetic support for a two-step mechanism where phosphoryl transfer from ATP to glutamate precedes reaction with ammonia.

Adenosine Triphosphatase from Rat Liver Mitochondria: II. INTERACTION WITH ADENOSINE DIPHOSPHATE

Abstract Homogeneous mitochondrial ATPase from rat liver binds ADP in a rapidly reversible manner. The enzyme-bound ADP can be recovered quantitatively as ADP. In the absence of Mg2+, the enzyme exhibits 0.88 binding sites for ADP per enzyme molecule with an intrinsic dissociation constant of 0.94 µm. In the presence of Mg2+, the enzyme loses 90% of its ATPase activity but does not lose the ability to bind ADP. Short term binding experiments detect 0.65 binding sites per enzyme molecule with an intrinsic dissociation constant of 2.1 µm. The Km (ADP) for oxidative phosphorylation catalyzed by purified inner membrane vesicles, in which mitochondrial ATPase is located on the outer surface of the membrane directly available to added ADP, was found to be 3.8 µm. Aurovertin, a potent inhibitor of oxidative phosphorylation, inhibits binding of ADP by mitochondrial ATPase in the absence of Mg2+ in a manner similar to its inhibition of oxidative phosphorylation. In the presence of Mg2+, however, binding is enhanced by aurovertin. Taken together, the results show that the binding of ADP by soluble mitochondrial ATPase has important properties in common with the interaction of ADP with the functional oxidative phosphorylation system.