Elucidating Events within the Black Box of Enzyme Catalysis in Energy Metabolism: Insights into the Molecular Mechanism of ATP Hydrolysis by F1-ATPase

Abstract

Oxygen exchange reactions occurring at β-catalytic sites of the FOF1-ATP synthase/F1-ATPase imprint a unique record of molecular events during the catalytic cycle of ATP synthesis/hydrolysis. This work presents a new theory of oxygen exchange and tests it on oxygen exchange data recorded on ATP hydrolysis by mitochondrial F1-ATPase (MF1). The apparent rate constant of oxygen exchange governing the intermediate Pi–HOH exchange accompanying ATP hydrolysis is determined by kinetic analysis over a ~50,000-fold range of substrate ATP concentration (0.1–5000 μM) and a corresponding ~200-fold range of reaction velocity (3.5–650 [moles of Pi/{moles of F1-ATPase}−1 s−1]). Isotopomer distributions of [18O]Pi species containing 0, 1, 2, and 3 labeled oxygen atoms predicted by the theory have been quantified and shown to be in perfect agreement with the experimental distributions over the entire range of medium ATP concentrations without employing adjustable parameters. A novel molecular mechanism of steady-state multisite ATP hydrolysis by the F1-ATPase has been proposed. Our results show that steady-state ATP hydrolysis by F1-ATPase occurs with all three sites occupied by Mg-nucleotide. The various implications arising from models of energy coupling in ATP synthesis/hydrolysis by the ATP synthase/F1-ATPase have been discussed. Current models of ATP hydrolysis by F1-ATPase, including those postulated from single-molecule data, are shown to be effectively bisite models that contradict the data. The trisite catalysis formulated by Nath’s torsional mechanism of energy transduction and ATP synthesis/hydrolysis since its first appearance 25 years ago is shown to be in better accord with the experimental record. The total biochemical information on ATP hydrolysis is integrated into a consistent model by the torsional mechanism of ATP synthesis/hydrolysis and shown to elucidate the elementary chemical and mechanical events within the black box of enzyme catalysis in energy metabolism by F1-ATPase.

Phosphorus Chemistry at the Roots of Bioenergetics: Ligand Permutation as the Molecular Basis of the Mechanism of ATP Synthesis/Hydrolysis by FOF1-ATP Synthase

“…Figure 4 shows the results of our experiments on hydrolysis by mitochondrial F 1 on the [ 18 The presence of multiple sites of water entry in ATP hydrolysis depending on ATP (𝑛 = 3 for ATP >3-5 µM, and 𝑛 = 2 for ATP <3-5 µM) (Figure 4) readily explains the observations of the high rates of both the Pi-HOH exchange and the ATP-HOH exchange (with respect to the rate of the Pi-ATP exchange) discussed at length in Section 2.2.2, as also the fact that these rates were found to be above the theoretical limits derived in Section 2.2.1 in a number of carefully carried out experiments. The rapid intermediate Pi-HOH and ATP-HOH exchanges are also explained if the exchanges do not involve the overall dynamic reversal of, but instead occur because of the lack of, absolute spatial selectivity in the binding of oxygen atoms of tetrahedral phosphate to enzyme [57,79,91]. The presence of multiple sites of water entry in ATP hydrolysis depending on ATP (n = 3 for ATP > 3-5 µM, and n = 2 for ATP < 3-5 µM) (Figure 4) readily explains the observations of the high rates of both the Pi-HOH exchange and the ATP-HOH exchange (with respect to the rate of the Pi-ATP exchange) discussed at length in Section 2.2.2, as also the fact that these rates were found to be above the theoretical limits derived in Section 2.2.1 in a number of carefully carried out experiments.…”

“…The presence of multiple sites of water entry in ATP hydrolysis depending on ATP (n = 3 for ATP > 3-5 µM, and n = 2 for ATP < 3-5 µM) (Figure 4) readily explains the observations of the high rates of both the Pi-HOH exchange and the ATP-HOH exchange (with respect to the rate of the Pi-ATP exchange) discussed at length in Section 2.2.2, as also the fact that these rates were found to be above the theoretical limits derived in Section 2.2.1 in a number of carefully carried out experiments. The rapid intermediate Pi-HOH and ATP-HOH exchanges are also explained if the exchanges do not involve the overall dynamic reversal of, but instead occur because of the lack of, absolute spatial selectivity in the binding of oxygen atoms of tetrahedral phosphate to enzyme [57,79,91].…”

“…A general kinetic approach to oxygen exchange-that makes no specific assumptions about mechanism-has been developed for ATP synthesis [57], and most recently for the intermediate Pi-HOH exchange accompanying ATP hydrolysis [91]. A principal result of the latter treatment (Equation ( 6) in Ref.…”

“…A principal result of the latter treatment (Equation ( 6) in Ref. [91]) is reproduced here as Equation ( 5)…”

“…in which k is the average apparent rate of oxygen exchange per s of turnover time, t, per catalytic site. The expression for the amount of oxygen exchange, kt, is given by Equation ( 5) irrespective of whether the exchange occurred prior to the cleavage of bound ATP, at an intermediate stage of hydrolysis, or following hydrolysis due to exchange between the oxygen of tightly bound Pi and that of medium water [91]. Equation ( 5) also satisfactorily accounts for the initial conditions, and mathematically corrects for any 18 O of water already incorporated into a phosphate group [91].…”