Replacement of the F 0 F 1 ATP synthase ␥ subunit Met-23 with Lys (␥M23K) perturbs coupling efficiency between transport and catalysis (Shin, K., Nakamoto, R. K., Maeda, M., and Futai, M. (1992) J. Biol. Chem. 267, 20835-20839). We demonstrate here that the ␥M23K mutation causes altered interactions between subunits. Binding of ␦ or ⑀ subunits stabilizes the ␣ 3 ␤ 3 ␥ complex, which becomes destabilized by the mutation. Significantly, the inhibition of F 1 ATP hydrolysis by the ⑀ subunit is no longer relieved when the ␥M23K mutant F 1 is bound to F 0 . Steady state Arrhenius analysis reveals that the ␥M23K enzyme has increased activation energies for the catalytic transition state. These results suggest that the mutation causes the formation of additional bonds within the enzyme that must be broken in order to achieve the transition state. Based on the x-ray crystallographic structure of Abrahams et al. (Abrahams, J. P., Leslie, A. G. W., Lutter, R., and Walker, J. E. (1994) Nature 370, 621-628), the additional bond is likely due to ␥M23K forming an ionized hydrogen bond with one of the ␤Glu-381 residues. Two second site mutations, ␥Q269R and ␥R242C, suppress the effects of ␥M23K and decrease activation energies for the ␥M23K enzyme. We conclude that ␥M23K is an added function mutation that increases the energy of interaction between ␥ and ␤ subunits. The additional interaction perturbs transmission of conformational information such that ⑀ inhibition of ATPase activity is not relieved and coupling efficiency is lowered.The F 0 F 1 ATP synthase links two disparate functions: transport of protons across a membrane and catalysis of ATP synthesis or hydrolysis (for reviews see Refs. 1-5). The fully cooperative mechanism of ATP hydrolysis requires a minimum of three different subunits in a complex containing ␣ 3 ␤ 3 ␥ 1 . The transport mechanism is most likely assembled from F 0 sector subunits. In the Escherichia coli complex, transport requires three different membrane-spanning subunits, a 1 b 2 c ϳ10 (6). In addition, two more soluble subunits, ␦ and ⑀, are needed to reconstitute catalytic and transport sectors so that they are coupled to carry out ATP-driven proton pumping or ⌬ Hϩ -driven ATP synthesis (7-10).Catalysis and transport mechanisms most likely communicate indirectly through a series of conformational and electrostatic interactions. Conformational changes relevant to the catalytic state of the enzyme or the presence of a ⌬ H ϩ have been detected by several methods including altered cross-linking patterns, protease susceptibility, environmentally sensitive fluorescent probes, accessibility of epitopes, x-ray diffraction, cryoelectron microscopy, and spectroscopic analyses (reviewed in Refs. 11 and 12). High resolution structural information based on crystals of the bovine mitochondrial F 1 has also provided a great deal of information about possible subunit interactions that may be involved in linking transport and catalysis (13).Mutagenic analysis has also yielded important information about the coupl...

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.

The F0F1 ATP synthase is a large multisubunit complex that couples translocation of protons down an electrochemical gradient to the synthesis of ATP. Recent advances in structural analyses have led to the demonstration that the enzyme utilizes a rotational catalytic mechanism. Kinetic and biochemical evidence is consistent with the expected equal participation of the three catalytic sites in the alpha 3 beta 3 hexamer, which operate in sequential, cooperative reaction pathways. The rotation of the core gamma subunit plays critical roles in establishing the conformation of the sites and the cooperative interactions. Mutational analyses have shown that the rotor subunits are responsible for coupling and in doing so transmit specific conformational information between transport and catalysis.

(Re)Building a Kidney is a National Institute of Diabetes and Digestive and Kidney Diseases-led consortium to optimize approaches for the isolation, expansion, and differentiation of appropriate kidney cell types and the integration of these cells into complex structures that replicate human kidney function. The ultimate goals of the consortium are two-fold: to develop and implement strategies for engineering of replacement kidney tissue, and to devise strategies to stimulate regeneration of nephrons to restore failing kidney function. Projects within the consortium will answer fundamental questions regarding human gene expression in the developing kidney, essential signaling crosstalk between distinct cell types of the developing kidney, how to derive the many cell types of the kidney through directed differentiation of human pluripotent stem cells, which bioengineering or scaffolding strategies have the most potential for kidney tissue formation, and basic parameters of the regenerative response to injury. As these projects progress, the consortium will incorporate systematic investigations in physiologic function of and differentiated kidney tissue, strategies for engraftment in experimental animals, and development of therapeutic approaches to activate innate reparative responses.