We previously demonstrated that the Escherichia coli F0F1-ATP synthase mutation, γM23K, caused increased energy of interaction between γ- and β-subunits which was correlated to inefficient coupling between catalysis and transport [Al-Shawi, Ketchum and Nakamoto (1997) J. Biol. Chem. 272, 2300-2306]. Based on these results and the X-ray crystallographic structure of bovine F1-ATPase [Abrahams, Leslie, Lutter and Walker (1994) Nature (London) 370, 621-628] γM23K is believed to form an ionized hydrogen bond with βGlu-381 in the conserved β380DELSEED386 segment. In this report, we further test the role of γ-β-subunit interactions by introducing a series of substitutions for βGlu-381 and γArg-242, the residue which forms a hydrogen bond with βGlu-381 in the wild-type enzyme. βE381A, D, and Q were able to restore efficient coupling when co-expressed with γM23K. All three mutations reversed the increased transition state thermodynamic parameters for steady state ATP hydrolysis caused by γM23K. βE381K by itself caused inefficient coupling, but opposite from the effect of γM23K, the transition state thermodynamic parameters were lower than wild-type. These results suggest that the βE381K mutation perturbs the γ-β-subunit interaction and the local conformation of the β380DELSEED386 segment in a specific way that disrupts the communication of coupling information between transport and catalysis. βE381A, L, K, and R, and γR242L and E mutations perturbed enzyme assembly and stability to varying degrees. These results provide functional evidence that the β380DELSEED386 segment and its interactions with the γ-subunit are involved in the mechanism of coupling.
Role of conserved residues within helices IV and VIII of the oxaloacetate decarboxylase β subunit in the energy coupling mechanism of the Na+
pump
