Site-directed mutagenesis of stable adenosine triphosphate synthase

Abstract

Evidence was obtained that four ionizable residues in the α and β subunits of thermophilic ATP synthase (TF 0F 1), corresponding to Lys-21 and Asp-119 in the MgATP binding segments of adenylate kinase, are essential for the normal catalytic activity. T 0F 1 was used because it is the only ATP synthase whose α-, β- and γ-subunits can be reassembled into an active complex in the absence of both ATP and Mg. Lys-164 and Asp-252 of its β-subunit were modified to isoleucine and asparagine, respectively, by site-directed mutagenesis using a multifunctional plasmid, and these genes were over-expressed in Escherichia coli. The resulting βI164 and βN252 subunits were both noncatalytic after re-assembly into the αβγ-complex, even though both subunits bound significant amounts of ADP. When Lys-175 and Asp-261 of the α-subunit were similarly replaced by isoleucine and asparagine, respectively, the resulting αI175 subunit reassembled weakly into an oligomer, while the αN261 subunit showed an increased dissociation constant for ADP and was reconstituted into an αβγ-complex that showed no inter-subunit cooperativity.