Abstract
Replacement of the F0F1 ATP synthase gamma subunit Met-23 with Lys (gammaM23K) 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 gammaM23K mutation causes altered interactions between subunits. Binding of delta or epsilon subunits stabilizes the alpha3beta3gamma complex, which becomes destabilized by the mutation. Significantly, the inhibition of F1 ATP hydrolysis by the epsilon subunit is no longer relieved when the gammaM23K mutant F1 is bound to F0. Steady state Arrhenius analysis reveals that the gammaM23K 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 gammaM23K forming an ionized hydrogen bond with one of the betaGlu-381 residues. Two second site mutations, gammaQ269R and gammaR242C, suppress the effects of gammaM23K and decrease activation energies for the gammaM23K enzyme. We conclude that gammaM23K is an added function mutation that increases the energy of interaction between gamma and beta subunits. The additional interaction perturbs transmission of conformational information such that epsilon inhibition of ATPase activity is not relieved and coupling efficiency is lowered.
Highlights
Catalysis and transport mechanisms most likely communicate indirectly through a series of conformational and electrostatic interactions
Restoration of efficient coupling in the ␥Met-23 3 Lys (␥M23K) mutant enzyme was conferred by several second site mutations near the carboxyl terminus of the ␥ subunit including the replacement of amino acid ␥Arg-242 and seven different residues between ␥Gln-269 and ␥Ala-280 [20]
We provide evidence that the ␥M23K mutation is an added function mutation that increases the energy of interaction between ␥ and  subunits
Summary
Catalysis and transport mechanisms most likely communicate indirectly through a series of conformational and electrostatic interactions. The most clear example of these mutations is replacement of ␥Met-23 with Arg or Lys [19] These mutations caused greatly reduced ATP-dependent proton pumping and ATP synthesis rates without strongly affecting catalytic or transport functions. Restoration of efficient coupling in the ␥Met-23 3 Lys (␥M23K) mutant enzyme was conferred by several second site mutations near the carboxyl terminus of the ␥ subunit including the replacement of amino acid ␥Arg-242 and seven different residues between ␥Gln-269 and ␥Ala-280 [20]. We provide evidence that the ␥M23K mutation is an added function mutation that increases the energy of interaction between ␥ and  subunits The consequences of this increase are destabilization of the ␣33␥ complex and inefficient coupling between transport and catalysis. The suppressor mutations described above counteract by decreasing the energy of interaction
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