Catalytic and Structural Importance of Gly-454, Tyr-455, and Leu-456 in the Carboxy-Terminal Region ofEscherichia coliF1-ATPase α Subunit

Monoclonal antibody α110 recognizes Leu-456 in the α subunit of theEscherichia coliF1-ATPase. Binding of this antibody to the α subunit or mutation of this residue to Pro caused enhancement of the ATPase activity, suggesting that this residue is involved in the catalytic mechanism of this molecule (H. Kanazawaet al.(1995)Arch. Biochem. Biophys.317, 348–356). Leu-456 together with Gly-454 and Tyr-455 are the only residues in the carboxy-terminal 75 amino acids conserved among various species, suggesting that these three residues play important roles in catalysis by the ATPase. Here, we introduced site-directed mutations into these residues. Not only L456P but also G454L, Y455K, Y455L, and L456N mutations caused enhancement of the ATPase activity. Surprisingly, Y455V, L456H, and L456S caused assembly defects of F1subunits on the membrane. Reconstitution of the αβγ complex from the wild-type β and γ subunits with the mutant α subunit (L456P) exhibited enhanced ATPase activity. Addition of δ or ϵ fused to glutathioneS-transferase which are functionally similar to the δ and ϵ subunits, respectively, to the reconstituted F1-ATPase did not cause significant enhancement of its activity. Decreased interaction between α and β subunits with the L456P mutation was detected by the yeast two-hybrid system. According to the deduced three-dimensional structure of the bovine α subunit, Leu-456, Gly-454, and Tyr-455 are included in a small α helix. These results suggest that this α helix affects interaction of the α subunit with the β subunit but not with δ or ϵ, which may be important for the catalytic mechanism and F1assembly.