Monitoring ATP hydrolysis and ATPase inhibitor screening using (1)H NMR.
Ontology highlight
ABSTRACT:
SUBMITTER: Guo B
PROVIDER: S-EPMC4262093 | biostudies-literature | 2014 Oct
REPOSITORIES: biostudies-literature
Publications
Monitoring ATP hydrolysis and ATPase inhibitor screening using (1)H NMR.
Guo Bingqian B Gurel Pinar S PS Shu Rui R Higgs Henry N HN Pellegrini Maria M Mierke Dale F DF
Chemical communications (Cambridge, England) 20141001 81
We present a versatile method to characterize ATPase and kinase activities and discover new inhibitors of these proteins. The proton NMR-based assay directly monitors ATP turnover and is easy to implement, requires no additional reagents and can potentially be applied to GTP. We validated the method's accuracy, applied it to the monitoring of ATP turnover by actin and to the screening of ATPase inhibitors, and showed that it is also applicable for the monitoring of GTP hydrolysis. ...[more]
Similar Datasets
Project description:Most of the cellular ATP in living organisms is synthesized by the enzyme F(1)F(o)-ATP synthase. The water soluble F(1) part of the enzyme can also work in reverse and utilize the chemical energy released during ATP hydrolysis to generate mechanical motion. Despite the availability of a large amount of biochemical data and several x-ray crystallographic structures of F(1), there still remains a considerable lack of understanding as to how this protein efficiently converts the chemical energy released during the reaction ATP + H(2)O --> ADP + P(i) into mechanical motion of the stalk. We report here an ab initio QM/MM study of ATP hydrolysis in the beta(TP) catalytic site of F(1). Our simulations provide an atomic level description of the reaction path, its energetics, and the interaction of the nucleotide with the protein environment during catalysis. The simulations suggest that the reaction path with the lowest potential energy barrier proceeds via nucleophilic attack on the gamma-phosphate involving two water molecules. Furthermore, the ATP hydrolysis reaction in beta(TP) is found to be endothermic, demonstrating that the catalytic site is able to support the synthesis of ATP and does not promote ATP hydrolysis in the particular conformation studied.
| S-EPMC1303451 | biostudies-literature
Project description:The enzyme F1-adenosine triphosphatase (ATPase) is a molecular motor that converts the chemical energy stored in the molecule adenosine triphosphate (ATP) into mechanical rotation of its gamma-subunit. During steady-state catalysis, the three catalytic sites of F1 operate in a cooperative fashion such that at every instant each site is in a different conformation corresponding to a different stage along the catalytic cycle. Notwithstanding a large amount of biochemical and, recently, structural data, we still lack an understanding of how ATP hydrolysis in F1 is coupled to mechanical motion and how the catalytic sites achieve cooperativity during rotatory catalysis. In this publication, we report combined quantum mechanical/molecular mechanical simulations of ATP hydrolysis in the betaTP and betaDP catalytic sites of F1-ATPase. Our simulations reveal a dramatic change in the reaction energetics from strongly endothermic in betaTP to approximately equienergetic in betaDP. The simulations identify the responsible protein residues, the arginine finger alphaR373 being the most important one. Similar to our earlier study of betaTP, we find a multicenter proton relay mechanism to be the energetically most favorable hydrolysis pathway. The results elucidate how cooperativity between catalytic sites might be achieved by this remarkable molecular motor.
| S-EPMC1304769 | biostudies-literature
Project description:Vacuolar-type H(+)-ATPase (V-ATPase) catalyzes ATP synthesis and hydrolysis coupled with proton translocation across membranes via a rotary motor mechanism. Here we report biochemical and biophysical catalytic properties of V-ATPase from Thermus thermophilus. ATP hydrolysis of V-ATPase was severely inhibited by entrapment of Mg-ADP in the catalytic site. In contrast, the enzyme was very active for ATP synthesis (approximately 70 s(-1)) with the K(m) values for ADP and phosphate being 4.7 +/- 0.5 and 460 +/- 30 microm, respectively. Single molecule observation showed V-ATPase rotated in a 120 degrees stepwise manner, and analysis of dwelling time allowed the binding rate constant k(on) for ATP to be estimated ( approximately 1.1 x 10(6) m(-1) s(-1)), which was much lower than the k(on) (= V(max)/K(m)) for ADP ( approximately 1.4 x 10(7) m(-1) s(-1)). The slower k(on)(ATP) than k(on)(ADP) and strong Mg-ADP inhibition may contribute to prevent wasteful consumption of ATP under in vivo conditions when the proton motive force collapses.
| S-EPMC3258951 | biostudies-literature
Project description:GTPases perform a wide range of functions, ranging from protein synthesis to cell signaling. Of all known GTPases, only eight are conserved across all three domains of life. YchF is one of these eight universally conserved GTPases; however, its cellular function and enzymatic properties are poorly understood. YchF differs from the classical GTPases in that it has a higher affinity for ATP than for GTP and is a functional ATPase. As a hydrophobic amino acid-substituted ATPase, YchF does not possess the canonical catalytic Gln required for nucleotide hydrolysis. To elucidate the catalytic mechanism of ATP hydrolysis by YchF, we have taken a two-pronged approach combining classical biochemical and in silico techniques. The use of molecular dynamics simulations allowed us to complement our biochemical findings with information about the structural dynamics of YchF. We have thereby identified the highly conserved His-114 as critical for the ATPase activity of YchF from Escherichia coli. His-114 is located in a flexible loop of the G-domain, which undergoes nucleotide-dependent conformational changes. The use of a catalytic His is also observed in the hydrophobic amino acid-substituted GTPase RbgA and is an identifier of the translational GTPase family.
| S-EPMC4513122 | biostudies-literature
Project description:It is largely unknown how the typical homomeric ring geometry of ATPases associated with various cellular activities enables them to perform mechanical work. Small-angle solution X-ray scattering, crystallography, and electron microscopy (EM) reconstructions revealed that partial ATP occupancy caused the heptameric closed ring of the bacterial enhancer-binding protein (bEBP) NtrC1 to rearrange into a hexameric split ring of striking asymmetry. The highly conserved and functionally crucial GAFTGA loops responsible for interacting with ?54-RNA polymerase formed a spiral staircase. We propose that splitting of the ensemble directs ATP hydrolysis within the oligomer, and the ring's asymmetry guides interaction between ATPase and the complex of ?54 and promoter DNA. Similarity between the structure of the transcriptional activator NtrC1 and those of distantly related helicases Rho and E1 reveals a general mechanism in homomeric ATPases whereby complex allostery within the ring geometry forms asymmetric functional states that allow these biological motors to exert directional forces on their target macromolecules.
| S-EPMC3841738 | biostudies-literature
Project description:One of the motive forces for F1-ATPase rotation is the conformational change of the catalytically active ? subunit due to closing and opening motions caused by ATP binding and hydrolysis, respectively. The closing motion is accomplished in two steps: the hydrogen-bond network around ATP changes and then the entire structure changes via B-helix sliding, as shown in our previous study. Here, we investigated the opening motion induced by ATP hydrolysis using all-atom free-energy simulations, combining the nudged elastic band method and umbrella sampling molecular-dynamics simulations. Because hydrolysis requires residues in the ? subunit, the simulations were performed with the ?? dimer. The results indicate that the large-scale opening motion is also achieved by the B-helix sliding (in the reverse direction). However, the sliding mechanism is different from that of ATP binding because sliding is triggered by separation of the hydrolysis products ADP and Pi. We also addressed several important issues: 1), the timing of the product Pi release; 2), the unresolved half-closed ? structure; and 3), the ADP release mechanism. These issues are fundamental for motor function; thus, the rotational mechanism of the entire F1-ATPase is also elucidated through this ?? study. During the conformational change, conserved residues among the ATPase proteins play important roles, suggesting that the obtained mechanism may be shared with other ATPase proteins. When combined with our previous studies, these results provide a comprehensive view of the ?-subunit conformational change that drives the ATPase.
| S-EPMC4286612 | biostudies-literature
Project description:The detailed mechanism of ATP hydrolysis in ATP-binding cassette (ABC) transporters is still not fully understood. Here, we employed 31P solid-state NMR to probe the conformational changes and dynamics during the catalytic cycle by locking the multidrug ABC transporter BmrA in prehydrolytic, transition, and posthydrolytic states, using a combination of mutants and ATP analogues. The 31P spectra reveal that ATP binds strongly in the prehydrolytic state to both ATP-binding sites as inferred from the analysis of the nonhydrolytic E504A mutant. In the transition state of wild-type BmrA, the symmetry of the dimer is broken and only a single site is tightly bound to ADP:Mg2+:vanadate, while the second site is more 'open' allowing exchange with the nucleotides in the solvent. In the posthydrolytic state, weak binding, as characterized by chemical exchange with free ADP and by asymmetric 31P-31P two-dimensional (2D) correlation spectra, is observed for both sites. Revisiting the 13C spectra in light of these findings confirms the conformational nonequivalence of the two nucleotide-binding sites in the transition state. Our results show that following ATP binding, the symmetry of the ATP-binding sites of BmrA is lost in the ATP-hydrolysis step, but is then recovered in the posthydrolytic ADP-bound state.
| S-EPMC9284561 | biostudies-literature
Project description:Although the binding change mechanism of rotary catalysis by which F1-ATPase hydrolyzes ATP has been supported by equilibrium, kinetic, and structural observations, many questions concerning the function remain unanswered. Because of the importance of this enzyme, the search for a full understanding of its mechanism is a key problem in structural biology. Making use of the results of free energy simulations and experimental binding constant measurements, a model is developed for the free energy change during the hydrolysis cycle. This model makes possible the development of a kinetic scheme for ATP hydrolysis by F1-ATPase, in which the rate constants are associated with specific configurations of the beta subunits. An essential new element is that the strong binding site for ADP,Pi is shown to be the betaDP site, in contrast to the strong binding site for ATP, which is betaTP. This result provides a rationale for the rotation of the gamma subunit, which induces the cooperativity required for a tri-site binding change mechanism. The model explains a series of experimental data, including the ATP concentration dependence of the rate of hydrolysis and catalytic site occupation for both the Escherichia coli F1-ATPase (EcF1) and Thermophilic Bacillus PS3 F1-ATPase (TF1), which have different behavior.
| S-EPMC208758 | biostudies-literature
Project description:Hsp70 escort proteins (Hep) have been implicated as essential for maintaining the function of yeast mitochondrial hsp70 molecular chaperones (mtHsp70), but the role that escort proteins play in regulating mammalian chaperone folding and function has not been established. We present evidence that human mtHsp70 exhibits limited solubility due to aggregation mediated by its ATPase domain and show that human Hep directly enhances chaperone solubility through interactions with this domain. In the absence of Hep, mtHsp70 was insoluble when expressed in Escherichia coli, as was its isolated ATPase domain and a chimera having this domain fused to the peptide-binding domain of HscA, a soluble monomeric chaperone. In contrast, these proteins all exhibited increased solubility when expressed in the presence of Hep. In vitro studies further revealed that purified Hep regulates the interaction of mtHsp70 with nucleotides. Full-length mtHsp70 exhibited slow intrinsic ATP hydrolysis activity (6.8+/-0.2 x 10(-4) s(-1)) at 25 degrees C, which was stimulated up to 49-fold by Hep. Hep also stimulated the activity of the isolated ATPase domain, albeit to a lower maximal extent (11.5-fold). In addition, gel-filtration studies showed that formation of chaperone-escort protein complexes inhibited mtHsp70 self-association, and they revealed that Hep binding to full-length mtHsp70 and its isolated ATPase domain is strongest in the absence of nucleotides. These findings provide evidence that metazoan escort proteins regulate the catalytic activity and solubility of their cognate chaperones, and they indicate that both forms of regulation arise from interactions with the mtHsp70 ATPase domain.
| S-EPMC3258852 | biostudies-literature
Project description:The hexameric MoxR AAA+ ATPase RavA and the decameric lysine decarboxylase LdcI form a 3.3 MDa cage, proposed to assist assembly of specific respiratory complexes in E. coli. Here, we show that inside the LdcI-RavA cage, RavA hexamers adopt an asymmetric spiral conformation in which the nucleotide-free seam is constrained to two opposite orientations. Cryo-EM reconstructions of free RavA reveal two co-existing structural states: an asymmetric spiral, and a flat C2-symmetric closed ring characterised by two nucleotide-free seams. The closed ring RavA state bears close structural similarity to the pseudo two-fold symmetric crystal structure of the AAA+ unfoldase ClpX, suggesting a common ATPase mechanism. Based on these structures, and in light of the current knowledge regarding AAA+ ATPases, we propose different scenarios for the ATP hydrolysis cycle of free RavA and the LdcI-RavA cage-like complex, and extend the comparison to other AAA+ ATPases of clade 7.
| S-EPMC6987120 | biostudies-literature