Browse
Submit Data
Databases
API
Help

Dataset Information

0 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Properties of F1-ATPase from the uncD412 mutant of Escherichia coli.

ABSTRACT: Properties of purified F1-ATPase from Escherichia coli mutant strain AN484 (uncD412) have been studied in an attempt to understand why the amino acid substitution in the beta-subunit of this enzyme causes a tenfold reduction from normal MgATP hydrolysis rate. In most properties that were studied, uncD412 F1-ATPase resembled normal E. coli F1-ATPase. Both enzymes were found to contain a total of six adenine-nucleotide-binding sites, of which three were found to be non-exchangeable and three were exchangeable (catalytic) sites. Binding of the non-hydrolysable substrate analogue adenosine 5'-[beta gamma-imido]triphosphate (p[NH]ppA) to the three exchangeable sites showed apparent negative co-operativity. The binding affinities for p[NH]ppA, and also ADP, at the exchangeable sites were similar in the two enzymes. Both enzymes were inhibited by efrapeptin, aurovertin and p[NH]ppA, and were inactivated by dicyclohexylcarbodi-imide, 4-chloro-7-nitrobenzofurazan and p-fluorosulphonyl-benzoyl-5'-adenosine. Km values for CaATP and MgATP were similar in the two enzymes. uncD412 F1-ATPase was abnormally unstable at high pH, and dissociated into subunits readily with consequent loss of activity. The reason for the impairment of catalysis in uncD412 F1-ATPase cannot be stated with certainty from these studies. However we discuss the possibility that the mutation interrupts subunit interaction, thereby causing a partial impairment in the site-site co-operativity which is required for 'promotion' of catalysis in this enzyme.

SUBMITTER: Wise JG

PROVIDER: S-EPMC1152402 | biostudies-other | 1983 Nov

REPOSITORIES: biostudies-other

ACCESS DATA

Json Xml

Similar Datasets

Oxidative phosphorylation in Escherichia coli. Characterization of mutant strains in which F1-ATPase contains abnormal beta-subunits.

Project description:To facilitate study of the role of the beta-subunit in the membrane-bound proton-translocating ATPase of Escherichia coli, we identified mutant strains from which an F1-ATPase containing abnormal beta-subunits can be purified. Seventeen strains of E. coli, characterized by genetic complementation tests as carrying mutations in the uncD gene (which codes for the beta-subunit), were studied. The majority of these strains (11) were judged to be not useful, as their membranes lacked ATPase activity, and were either proton-permeable as prepared or remained proton-impermeable after washing with buffer of low ionic strength. A further two strains were of a type not hitherto reported, in that their membranes had ATPase activity, were proton-impermeable as prepared, and were not rendered proton-permeable by washing in buffer of low ionic strength. Presumably in these two strains F1-ATPase is not released in soluble form by this procedure. F1-ATPase of normal molecular size were purified from strains AN1340 (uncD478), AN937 (uncD430), AN938 (uncD431) and AN1543 (uncD484). F1-ATPase from strain AN1340 (uncD478) had 15% of normal specific Mg-dependent ATPase activity and 22% of normal ATP-synthesis activity. The F1-ATPase preparations from strains AN937, AN938 and AN1543 had respectively 1.7%, 1.8% and 0.2% of normal specific Mg-dependent ATPase activity, and each of these preparations had very low ATP-synthesis activity. The yield of F1-ATPase from the four strains described was almost twice that obtained from a normal haploid strain. The kinetics of Ca-dependent ATPase activity were unusual in each of the four F1-ATPase preparations. It is likely that these four mutant uncD F1-ATPase preparations will prove valuable for further experimental study of the F1-ATPase catalytic mechanism.

| S-EPMC1154237 | biostudies-other

Loss of protection by nucleotides against proteolysis and thiol modification in the isolated alpha-subunit from F1 ATPase of Escherichia coli mutant uncA401.

Project description:Binding of nucleotides to the high-affinity site of the isolated alpha subunit of normal Escherichia coli F1 adenosine triphosphatase (ATPase) results in partial protection against digestion by trypsin [Senda, Kanazawa, Tsuchiya & Futai (1983) Arch. Biochem. Biophys. 220, 398-440]. In contrast, the isolated alpha subunit from the defective ATPase of the E. coli uncA401 mutant (strain AN120) is cleaved by trypsin to peptides of less than 8000 Da in the presence of ADP or ATP (2.5 microM-110 mM). The nucleotide-dependent accessibility of thiol groups of the isolated alpha subunit was also studied. Two out of four thiol groups of the alpha subunit from normal ATPase are labelled by fluorescent maleimides or iodoacetates, but in the presence of ADP or ATP (0.14-1.2 mM), reaction of thiol groups with these labels is almost absent. Mutant alpha subunit, however, is labelled by these reagents at all four thiol groups in the presence or absence of ADP or ATP (1 mM). These results suggest that the mutation in the ATPase of strain AN120 leads either to the loss of the high-affinity nucleotide-binding site or affects transmission of allosteric changes that occur on binding of nucleotide to the isolated alpha subunit.

| S-EPMC1144407 | biostudies-other

Random mutagenesis of the gene for the beta-subunit of F1-ATPase from Escherichia coli.

Project description:ATP synthesis by oxidative phosphorylation in Escherichia coli occurs in catalytic sites on the beta-subunits of F1-ATPase. Random mutagenesis of the beta-subunit combined with phenotypic screening is potentially important for studies of the catalytic mechanism. However, when applied to haploid strains, this approach is hampered by a preponderance of mutants in which assembly of F1-ATPase in vivo is defective, precluding enzyme purification. Here we mutagenized plasmids carrying the uncD (beta-subunit) gene with hydroxylamine or N-methyl-N'-nitro-N-nitrosoguanidine and isolated, by phenotypic screening and complementation tests, six plasmids carrying mutant uncD alleles. When the mutant plasmids were used to transform a suitable uncD- strain, assembly of F1-ATPase in vivo occurred in each case. Moreover, in one case (beta Gly-223----Asp) F1-ATPase assembly proceeded although it had previously been reported that this mutation, when present on the chromosome of a haploid strain, prevented assembly of the enzyme in vivo. Therefore, this work demonstrates an improved approach for random mutagenesis of the F1-beta-subunit. Six new mutant uncD alleles were identified: beta Cys-137----Tyr; beta Gly-142----Asp; beta Gly-146----Ser; beta Gly-207----Asp; beta-Gly-223----Asp; and a double mutant beta Pro-403----Ser,Gly-415----Asp which we could not separate. The first five of these lie within or very close to the predicted catalytic nucleotide-binding domain of the beta-subunit. The double mutant lies outside this domain; we speculate that the region around residues beta 403-415 is part of an alpha-beta intersubunit contact surface. Membrane ATPase and ATP-driven proton pumping activities were impaired by all six mutations. Purified F1-ATPase was obtained from each mutant and shown to have impaired specific ATPase activity.

| S-EPMC1138526 | biostudies-other

Chaperones of F1-ATPase.

Project description:Mitochondrial F(1)-ATPase contains a hexamer of alternating alpha and beta subunits. The assembly of this structure requires two specialized chaperones, Atp11p and Atp12p, that bind transiently to beta and alpha. In the absence of Atp11p and Atp12p, the hexamer is not formed, and alpha and beta precipitate as large insoluble aggregates. An early model for the mechanism of chaperone-mediated F(1) assembly (Wang, Z. G., Sheluho, D., Gatti, D. L., and Ackerman, S. H. (2000) EMBO J. 19, 1486-1493) hypothesized that the chaperones themselves look very much like the alpha and beta subunits, and proposed an exchange of Atp11p for alpha and of Atp12p for beta; the driving force for the exchange was expected to be a higher affinity of alpha and beta for each other than for the respective chaperone partners. One important feature of this model was the prediction that as long as Atp11p is bound to beta and Atp12p is bound to alpha, the two F(1) subunits cannot interact at either the catalytic site or the noncatalytic site interface. Here we present the structures of Atp11p from Candida glabrata and Atp12p from Paracoccus denitrificans, and we show that some features of the Wang model are correct, namely that binding of the chaperones to alpha and beta prevents further interactions between these F(1) subunits. However, Atp11p and Atp12p do not resemble alpha or beta, and it is instead the F(1) gamma subunit that initiates the release of the chaperones from alpha and beta and their further assembly into the mature complex.

| S-EPMC2719352 | biostudies-literature

Crystal structures of mutant forms of the yeast F1 ATPase reveal two modes of uncoupling.

Project description:The mitochondrial ATP synthase couples the flow of protons with the phosphorylation of ADP. A class of mutations, the mitochondrial genome integrity (mgi) mutations, has been shown to uncouple this process in the yeast mitochondrial ATP synthase. Four mutant forms of the yeast F(1) ATPase with mgi mutations were crystallized; the structures were solved and analyzed. The analysis identifies two mechanisms of structural uncoupling: one in which the empty catalytic site is altered and in doing so, apparently disrupts substrate (phosphate) binding, and a second where the steric hindrance predicted between γLeu83 and β(DP) residues, Leu-391 and Glu-395, located in Catch 2 region, is reduced allowing rotation of the γ-subunit with less impedance. Overall, the structures provide key insights into the critical interactions in the yeast ATP synthase involved in the coupling process.

| S-EPMC2978584 | biostudies-literature

Anatomy of F1-ATPase powered rotation.

Project description:F1-ATPase, the catalytic complex of the ATP synthase, is a molecular motor that can consume ATP to drive rotation of the ?-subunit inside the ring of three ??-subunit heterodimers in 120° power strokes. To elucidate the mechanism of ATPase-powered rotation, we determined the angular velocity as a function of rotational position from single-molecule data collected at 200,000 frames per second with unprecedented signal-to-noise. Power stroke rotation is more complex than previously understood. This paper reports the unexpected discovery that a series of angular accelerations and decelerations occur during the power stroke. The decreases in angular velocity that occurred with the lower-affinity substrate ITP, which could not be explained by an increase in substrate-binding dwells, provides direct evidence that rotation depends on substrate binding affinity. The presence of elevated ADP concentrations not only increased dwells at 35° from the catalytic dwell consistent with competitive product inhibition but also decreased the angular velocity from 85° to 120°, indicating that ADP can remain bound to the catalytic site where product release occurs for the duration of the power stroke. The angular velocity profile also supports a model in which rotation is powered by Van der Waals repulsive forces during the final 85° of rotation, consistent with a transition from F1 structures 2HLD1 and 1H8E (Protein Data Bank).

| S-EPMC3956197 | biostudies-literature

F1-ATPase of Escherichia coli: the ?- inhibited state forms after ATP hydrolysis, is distinct from the ADP-inhibited state, and responds dynamically to catalytic site ligands.

Project description:F1-ATPase is the catalytic complex of rotary nanomotor ATP synthases. Bacterial ATP synthases can be autoinhibited by the C-terminal domain of subunit ?, which partially inserts into the enzyme's central rotor cavity to block functional subunit rotation. Using a kinetic, optical assay of F1·? binding and dissociation, we show that formation of the extended, inhibitory conformation of ? (?X) initiates after ATP hydrolysis at the catalytic dwell step. Prehydrolysis conditions prevent formation of the ?X state, and post-hydrolysis conditions stabilize it. We also show that ? inhibition and ADP inhibition are distinct, competing processes that can follow the catalytic dwell. We show that the N-terminal domain of ? is responsible for initial binding to F1 and provides most of the binding energy. Without the C-terminal domain, partial inhibition by the ? N-terminal domain is due to enhanced ADP inhibition. The rapid effects of catalytic site ligands on conformational changes of F1-bound ? suggest dynamic conformational and rotational mobility in F1 that is paused near the catalytic dwell position.

| S-EPMC3611008 | biostudies-literature

Torsional elasticity and energetics of F1-ATPase.

Project description:F(o)F(1)-ATPase is a rotary motor protein synthesizing ATP from ADP driven by a cross-membrane proton gradient. The proton flow through the membrane-embedded F(o) generates the rotary torque that drives the rotation of the asymmetric shaft of F(1). Mechanical energy of the rotating shaft is used by the F(1) catalytic subunit to synthesize ATP. It was suggested that elastic power transmission with transient storage of energy in some compliant part of the shaft is required for the observed high turnover rate. We used atomistic simulations to study the spatial distribution and structural determinants of the F(1) torsional elasticity at the molecular level and to comprehensively characterize the elastic properties of F(1)-ATPase. Our fluctuation analysis revealed an unexpected heterogeneity of the F(1) shaft elasticity. Further, we found that the measured overall torsional moduli of the shaft arise from two distinct contributions, the intrinsic elasticity and the effective potential imposed on the shaft by the catalytic subunit. Separation of these two contributions provided a quantitative description of the coupling between the rotor and the catalytic subunit. This description enabled us to propose a minimal quantitative model of the F(1) energetics along the rotary degrees of freedom near the resting state observed in the crystal structures. As opposed to the usually employed models where the motor mechanical progression is described by a single angular variable, our multidimensional treatment incorporates the spatially inhomogeneous nature of the shaft and its interactions with the stator and offers new insight into the mechanoenzymatics of F(1)-ATPase.

| S-EPMC3088579 | biostudies-literature

Temperature-sensitive reaction intermediate of F1-ATPase.

Project description:F(1)-ATPase is a rotary molecular motor that makes 120 degrees stepping rotations, with each step being driven by a single-ATP hydrolysis. In this study, a new reaction intermediate of F(1)-ATPase was discovered at a temperature below 4 degrees C, which makes a pause at the same angle in its rotation as when ATP binds. The rate constant of the intermediate reaction was strongly dependent on temperature with a Q(10) factor of 19, implying that the intermediate reaction accompanies a large conformational change. Kinetic analyses showed that the intermediate state does not correspond to ATP binding or hydrolysis. The addition of ADP to the reaction mixture did not alter the angular position of the intermediate state, but specifically lengthened the time constant of this state. Conversely, the addition of inorganic phosphate caused a pause at an angle of +80 degrees from that of the intermediate state. These observations strongly suggest that the newly found reaction intermediate is an ADP-releasing step.

| S-EPMC2246616 | biostudies-literature

Changes in the adenine nucleotide and inorganic phosphate content of Escherichia coli F1-ATPase during ATP synthesis in dimethyl sulphoxide.

Project description:Escherichia coli F1-ATPase contained 2.9 +/- 0.1 mol of adenine nucleotide and 3.1 +/- 0.3 mol of Pi/mol of enzyme. After preincubation with ATP, the nucleotide and phosphate contents were 5.6 and 6.0 +/- 0.5 mol/mol of enzyme respectively. The F1-ATPase was induced to synthesize ATP in the presence of 30% (v/v) dimethyl sulphoxide (Me2SO). The ATP originated from endogenous bound ADP. The bound adenine nucleotide and Pi contents of the enzyme during the time course of ATP synthesis were investigated by using F1-ATPase which had been preincubated with ATP. We show that the process of ATP synthesis in Me2SO involves (i) an initial rapid loss of nucleotide from the enzyme, the process being facilitated by exogenous Pi, (ii) a rapid loss of Pi from the enzyme, at least in the absence of exogenous Pi, (iii) re-binding of a portion of the lost nucleotide, and (iv) synthesis of ATP from bound ADP and exogenous Pi. It is proposed that transfer of the F1-ATPase to the Me2SO medium induces a change in the conformation of the enzyme to a form favouring ATP synthesis.

| S-EPMC1132940 | biostudies-other

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data