The Escherichia coli F_OF₁ γM23K Uncoupling Mutant Has a Higher K_0.5 for P_i. Transition State Analysis of This Mutant and Others Reveals That Synthesis and Hydrolysis Utilize the Same Kinetic Pathway

Abstract

The Escherichia coli FOF1 ATP synthase uncoupling mutation, gammaM23K, was found to increase the energy of interaction between gamma and beta subunits, prevent the proper utilization of binding energy to drive catalysis, and block the enzyme in a Pi release mode. In this paper, the effects of this mutation on substrate binding in cooperative ATP synthesis are assessed. Activation of ATP synthesis by ADP and Pi was determined for the gammaM23K FOF1. The K0.5 for ADP was not affected, but K0.5 for Pi was approximately 7-fold higher even though the apparent Vmax was close to the wild-type level. Wild-type enzyme had a turnover number of 82 s-1 at pH 7.5 and 30 degrees C. During oxidative phosphorylation, the apparent dissociation constant (KI) for ATP was not affected and was 5-6 mM for both wild-type and gammaM23K enzymes. Thus, the apparent binding affinity for ATP in the presence of DeltamuH+ was lowered by 7 orders of magnitude from the affinity measured at the high-affinity catalytic site. Arrhenius analysis of ATP synthesis for the gammaM23K FOF1 revealed that, like those of ATP hydrolysis, the transition state DeltaH was much more positive and TDeltaS was much less negative, adding up to little change in DeltaG. These results suggested that ATP synthesis is inefficient because of an extra bond between gamma and beta subunits which must be broken to achieve the transition state. Analysis of the transition state structures using isokinetic plots demonstrate that ATP hydrolysis and synthesis utilize the same kinetic pathway. Incorporating this information into a model for rotational catalysis suggests that at saturating substrate concentrations, the rate-limiting step for hydrolysis and synthesis is the rotational power stroke where each of the beta subunits changes conformation and affinity for nucleotide.

Intragenic and intergenic suppression of the Escherichia coli ATP synthase subunit a mutation of Gly-213 to Asn: functional interactions between residues in the proton transport site

“…For a given reaction series that uses the same transition state there should be a quantitative relationship between enthalpy and entropy of activation, such as an isokinetic relationship [54]. We previously demonstrated that the transitionstate thermodynamic parameters from ATP synthases with a variety of mutations or from different sources could be fitted to an isokinetic line [43,45] such as that shown in Figure 5. In isokinetic relationships there is a linear free-energy relationship between two rate constants (k " and k # ) measured at two temperatures (T # T " ), which can be given by the equation : log k # l ajb log k " where a and b are constants [54].…”

“…The conditions for the ACMA assay were optimized for maximal sensitivity and are detailed in the legend to Figure 3. ATP hydrolysis rates were determined at 30 mC in a buffer containing 50 mM Hepes\KOH, 10 mM ATP, 5 mM MgSO % and 1 µM carbonyl cyanide mchlorophenylhydrazone (CCCP), pH 7.5, with 5 mM phosphoenolpyruvate, 50 µg\ml pyruvate kinase and 0.1-0.3 mg\ml membrane protein [45]. For the Arrhenius analysis, the pH was adjusted to 7.5 at the appropriate temperature [43].…”

“…For the Arrhenius analysis, the pH was adjusted to 7.5 at the appropriate temperature [43]. ATP synthesis rates were determined at 30 mC as described previously [45]. The concentration of free Mg# + and MgATP# − was calculated by using the algorithm of Fabiato and Fabiato [46].…”

“…In spite of the intrinsic proton leak, the electron transport chain has excess capacity and is able to drive the maximal electrochemical gradient of protons, unless there is an additional leak [45]. Because the aG213N mutant strain was capable of oxidative phosphorylation-dependent growth, the mutant enzyme must have had at least a low level of activity.…”

“…We have previously used Arrhenius analyses of the catalytic transition state of steady-state ATP hydrolysis to assess the effects of amino acid replacements in the F " sector on the catalytic mechanism [42,43,45,49]. These analyses have been especially revealing with respect to the functional and physical interactions between the rotor γ subunit and the stator α and β subunits as well as an amino acid change at the γ-c subunit interface, γE208K, that affected coupling efficiency [49].…”