ENHANCEMENT OF ESCHERICHIA-COLI H-ATPASE CAUSED BY BINDING OF MONOCLONAL-ANTIBODIES IS ATTRIBUTED TO STRUCTURAL-CHANGES OF LEU-456 AND SER-440 IN THE ALPHA-SUBUNIT()

Five monoclonal antibodies against the alpha subunit of F-1-ATPase fro m Escherichia coli alpha 104, alpha 105, alpha 107, alpha 109, and alp ha 110 were prepared. The monoclonal antibodies alpha 104 and alpha 11 0 enhanced the F-1-ATPase activity maximally to 1.6- and 1.7-fold that of the wild-type, respectively, while alpha 105 did not. Both antibod ies bound to a peptide corresponding to the region between residues 35 4 and 513. Mutations in this region which caused reduced binding of th e alpha subunit to the antibodies were identified at residues Ser-440, Leu-456, Leu-471, Leu-482, Met-483, and Ser-506 for alpha 104 and res idues Ser-440, Leu-456, Leu-471, Asp-476, Leu-482, Met-483, and Ser-50 6 for alpha 110. These residues are possibly involved in the epitopes for the antibodies and are located close together on the surface of th e alpha subunit. Among the mutations, Leu-456 to Pro and Ser-440 to Pr o mutations caused increase of the F-1-ATPase activity up to 1.9 and 1 .2 times that of the wild-type, respectively, while Leu-471 to Pro mut ation caused a defect in assembly of the F-1-ATPase on the membrane. T he other mutations caused no significant change in ATPase activity. Th ese results suggested that Ser-440 and Leu-456 have an important role in regulating catalysis by the F-1-ATPase, but that the neighboring re sidue Leu-471 has an important role in assembly of the F-1-ATPase comp lex. It was also suggested that binding of the monoclonal antibodies a lpha 104 and alpha 110 to residues Ser-440 and Leu-456 caused local co nformational changes, leading to enhancing effects on F-1-ATPase activ ity similar to the Ser-440 to Pro and Leu-456 to Pro mutations. (C) 19 95 Academic Press, Inc.