Mechanism of inhibition of bovine F1-ATPase by resveratrol and related polyphenols.
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ABSTRACT:
SUBMITTER: Gledhill JR
PROVIDER: S-EPMC1948022 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:High-resolution crystallographic studies of a number of inhibited forms of bovine F1-ATPase have identified four independent types of inhibitory site: the catalytic site, the aurovertin B-binding site, the efrapeptin-binding site and the site to which the natural inhibitor protein IF1 binds. Hitherto, the binding sites for other inhibitors, such as polyphenolic phytochemicals, non-peptidyl lipophilic cations and amphiphilic peptides, have remained undefined. By employing multiple inhibition analysis, we have identified the binding sites for these compounds. Several of them bind to the known inhibitory sites. The amphiphilic peptides melittin and synthetic analogues of the mitochondrial import pre-sequence of yeast cytochrome oxidase subunit IV appear to mimic the natural inhibitor protein, and the polyphenolic phytochemical inhibitors resveratrol and piceatannol compete for the aurovertin B-binding site (or sites). The non-peptidyl lipophilic cation rhodamine 6G acts at a separate unidentified site, indicating that there are at least five inhibitory sites in the F1-ATPase. Each of the above inhibitors has significantly different activity against the bacterial Bacillus PS3 alpha3beta3gamma subcomplex compared with that observed with bovine F1-ATPase. IF1 does not inhibit the bacterial enzyme, even in the absence of the epsilon-subunit. An understanding of these inhibitors may enable rational development of therapeutic agents to act as novel antibiotics against bacterial ATP synthases or for the treatment of several disorders linked to the regulation of the ATP synthase, including ischaemia-reperfusion injury and some cancers.
| S-EPMC1134879 | biostudies-literature
Project description:ATPase inhibitory factor 1 (IF1) is a mitochondrial regulatory protein that blocks ATP hydrolysis of F1-ATPase, by inserting its N-terminus into the rotor-stator interface of F1-ATPase. Although previous studies have proposed a two-step model for IF1-mediated inhibition, the underlying molecular mechanism remains unclear. Here, we analysed the kinetics of IF1-mediated inhibition under a wide range of [ATP]s and [IF1]s, using bovine mitochondrial IF1 and F1-ATPase. Typical hyperbolic curves of inhibition rates with [IF1]s were observed at all [ATP]s tested, suggesting a two-step mechanism: the initial association of IF1 to F1-ATPase and the locking process, where IF1 blocks rotation by inserting its N-terminus. The initial association was dependent on ATP. Considering two principal rotation dwells, binding dwell and catalytic dwell, in F1-ATPase, this result means that IF1 associates with F1-ATPase in the catalytic-waiting state. In contrast, the isomerization process to the locking state was almost independent of ATP, suggesting that it is also independent of the F1-ATPase state. Further, we investigated the role of Glu30 or Tyr33 of IF1 in the two-step mechanism. Kinetic analysis showed that Glu30 is involved in the isomerization, whereas Tyr33 contributes to the initial association. Based on these findings, we propose an IF1-mediated inhibition scheme.
| S-EPMC8457647 | biostudies-literature
Project description:F1-ATPase (F1) is an ATP-driven rotary motor in which the three catalytic ? subunits in the stator ring sequentially induce the unidirectional rotation of the rotary ? subunit. Many lines of evidence have revealed open-to-closed conformational transitions in the ? subunit that swing the C-terminal domain inward. This conformational transition causes a C-terminal protruding loop with conserved sequence DELSEED to push the ? subunit. Previous work, where all residues of DELSEED were substituted with glycine to disrupt the specific interaction with ? and introduce conformational flexibility, showed that F1 still rotated, but that the torque was halved, indicating a remarkable impact on torque transmission. In this study, we conducted a stall-and-release experiment on F1 with a glycine-substituted DELSEED loop to investigate the impact of the glycine substitution on torque transmission upon ATP binding and ATP hydrolysis. The mutant F1 showed a significantly reduced angle-dependent change in ATP affinity, whereas there was no change in the equilibrium for ATP hydrolysis. These findings indicate that the DELSEED loop is predominantly responsible for torque transmission upon ATP binding but not for that upon ATP hydrolysis.
| S-EPMC4375457 | biostudies-literature
Project description:Molecular machines fueled by NTP play pivotal roles in a wide range of cellular activities. One common feature among NTP-driven molecular machines is that NTP binding is a major force-generating step among the elementary reaction steps comprising NTP hydrolysis. To understand the mechanism in detail,in this study, we conducted a single-molecule rotation assay of the ATP-driven rotary motor protein F1-ATPase using uridine triphosphate (UTP) and a base-free nucleotide (ribose triphosphate) to investigate the impact of a pyrimidine base or base depletion on kinetics and force generation. Although the binding rates of UTP and ribose triphosphate were 10(3) and 10(6) times, respectively, slower than that of ATP, they supported rotation, generating torque comparable to that generated by ATP. Affinity change of F1 to UTP coupled with rotation was determined, and the results again were comparable to those for ATP, suggesting that F1 exerts torque upon the affinity change to UTP via rotation similar to ATP-driven rotation. Thus, the adenine-ring significantly enhances the binding rate, although it is not directly involved in force generation. Taking into account the findings from another study on F1 with mutated phosphate-binding residues, it was proposed that progressive bond formation between the phosphate region and catalytic residues is responsible for the rotation-coupled change in affinity.
| S-EPMC4119285 | biostudies-literature
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:F1-ATPase (F1) is the rotary motor protein fueled by ATP hydrolysis. Previous studies have suggested that three charged residues are indispensable for catalysis of F1 as follows: the P-loop lysine in the phosphate-binding loop, GXXXXGK(T/S); a glutamic acid that activates water molecules for nucleophilic attack on the ?-phosphate of ATP (general base); and an arginine directly contacting the ?-phosphate (arginine finger). These residues are well conserved among P-loop NTPases. In this study, we investigated the role of these charged residues in catalysis and torque generation by analyzing alanine-substituted mutants in the single-molecule rotation assay. Surprisingly, all mutants continuously drove rotary motion, even though the rotational velocity was at least 100,000 times slower than that of wild type. Thus, although these charged residues contribute to highly efficient catalysis, they are not indispensable to chemo-mechanical energy coupling, and the rotary catalysis mechanism of F1 is far more robust than previously thought.
| S-EPMC4094045 | biostudies-literature
Project description:MgADP inhibition, which is considered as a part of the regulatory system of ATP synthase, is a well-known process common to all F1-ATPases, a soluble component of ATP synthase. The entrapment of inhibitory MgADP at catalytic sites terminates catalysis. Regulation by the ? subunit is a common mechanism among F1-ATPases from bacteria and plants. The relationship between these two forms of regulatory mechanisms is obscure because it is difficult to distinguish which is active at a particular moment. Here, using F1-ATPase from Bacillus subtilis (BF1), which is strongly affected by MgADP inhibition, we can distinguish MgADP inhibition from regulation by the ? subunit. The ? subunit did not inhibit but activated BF1. We conclude that the ? subunit relieves BF1 from MgADP inhibition.
| S-EPMC3742539 | biostudies-literature
Project description:The reaction scheme of rotary catalysis and the torque generation mechanism of bovine mitochondrial F1 (bMF1) were studied in single-molecule experiments. Under ATP-saturated concentrations, high-speed imaging of a single 40-nm gold bead attached to the γ subunit of bMF1 showed 2 types of intervening pauses during the rotation that were discriminated by short dwell and long dwell. Using ATPγS as a slowly hydrolyzing ATP derivative as well as using a functional mutant βE188D with slowed ATP hydrolysis, the 2 pausing events were distinctively identified. Buffer-exchange experiments with a nonhydrolyzable analog (AMP-PNP) revealed that the long dwell corresponds to the catalytic dwell, that is, the waiting state for hydrolysis, while it remains elusive which catalytic state short pause represents. The angular position of catalytic dwell was determined to be at +80° from the ATP-binding angle, mostly consistent with other F1s. The position of short dwell was found at 50 to 60° from catalytic dwell, that is, +10 to 20° from the ATP-binding angle. This is a distinct difference from human mitochondrial F1, which also shows intervening dwell that probably corresponds to the short dwell of bMF1, at +65° from the binding pause. Furthermore, we conducted "stall-and-release" experiments with magnetic tweezers to reveal how the binding affinity and hydrolysis equilibrium are modulated by the γ rotation. Similar to thermophilic F1, bMF1 showed a strong exponential increase in ATP affinity, while the hydrolysis equilibrium did not change significantly. This indicates that the ATP binding process generates larger torque than the hydrolysis process.
| S-EPMC6983367 | biostudies-literature
Project description:The structure of bovine F1-ATPase inhibited with ADP and beryllium fluoride at 2.0 angstroms resolution contains two ADP.BeF3- complexes mimicking ATP, bound in the catalytic sites of the beta(TP) and beta(DP) subunits. Except for a 1 angstrom shift in the guanidinium of alphaArg373, the conformations of catalytic side chains are very similar in both sites. However, the ordered water molecule that carries out nucleophilic attack on the gamma-phosphate of ATP during hydrolysis is 2.6 angstroms from the beryllium in the beta(DP) subunit and 3.8 angstroms away in the beta(TP) subunit, strongly indicating that the beta(DP) subunit is the catalytically active conformation. In the structure of F1-ATPase with five bound ADP molecules (three in alpha-subunits, one each in the beta(TP) and beta(DP) subunits), which has also been determined, the conformation of alphaArg373 suggests that it senses the presence (or absence) of the gamma-phosphate of ATP. Two catalytic schemes are discussed concerning the various structures of bovine F1-ATPase.
| S-EPMC514953 | biostudies-literature
Project description:We briefly review our theoretical study on the rotation scheme of F1-ATPase. In the scheme, the key factor is the water entropy which has been shown to drive a variety of self-assembly processes in biological systems. We decompose the crystal structure of F1-ATPase into three sub-complexes each of which is composed of the ? subunit, one of the ? subunits, and two ? subunits adjacent to them. The ?E, ?TP, and ?DP subunits are involved in the sub-complexes I, II, and III, respectively. We calculate the hydration entropy of each sub-complex using a hybrid of the integral equation theory for molecular liquids and the morphometric approach. It is found that the absolute value of the hydration entropy follows the order, sub-complex I > sub-complex II > sub-complex III. Moreover, the differences are quite large, which manifests highly asymmetrical packing of F1-ATPase. In our picture, this asymmetrical packing plays crucially important roles in the rotation of the ? subunit. We discuss how the rotation is induced by the water-entropy effect coupled with such chemical processes as ATP binding, ATP hydrolysis, and release of the products.
| S-EPMC5036781 | biostudies-literature