INTERGENIC SUPPRESSION OF THE GAMMA-M23K UNCOUPLING MUTATION IN F0F1 ATP SYNTHASE BY BETA-GLU-381 SUBSTITUTIONS - THE ROLE OF THE BETA(DELSEED386)-D-380 SEGMENT IN ENERGY COUPLING

We previously demonstrated that the Escherichia coli F0F1-ATP synthase mutation, gamma M23K, caused increased energy of interaction between gamma- and beta-subunits which was correlated to inefficient coupling between catalysis and transport [Al-Shawl, Ketchum and Nakamoto (1997) J. Biol. Chem. 272, 2300-2306]. Based on these results and the X-ray crystallographic structure of bovine F-1-ATPase [Abrahams: Leslie, Lut ter and Walker (1994) Nature (London) 370, 621-628] gamma M23K is beli eved to form an ionized hydrogen bond with beta Glu-381 in the conserv ed beta(380)DELSEED(386) segment. In this report, we further test the role of gamma-beta-subunit interactions by introducing a series of sub stitutions for beta Glu-381 and gamma Arg-242, the residue which forms a hydrogen bond with beta Glu-381 in the wild-type enzyme. beta E381A , D, and Q were able to restore efficient coupling when coexpressed wi th gamma M23K. All three mutations reversed the increased transition s tate thermodynamic parameters for steady state ATP hydrolysis caused b y gamma M23K. beta E381K by itself caused inefficient coupling, but op posite from the effect of gamma M23K, the transition state thermodynam ic parameters were lower than wild-type. These results suggest that th e beta E381K mutation perturbs the gamma-beta-subunit interaction and the local conformation of the beta(380)DELSEED(386) segment in a speci fic way that disrupts the communication of coupling information betwee n transport and catalysis. beta E381A, L, K, and R, and gamma R242L an d E mutations perturbed enzyme assembly and stability to varying degre es. These results provide functional evidence that the beta(380)DELSEE D(386) segment and its interactions with the gamma-subunit are involve d in the mechanism of coupling.