Gene Structure and Function of Thermophilic ATP Synthase

Abstract

An operon for thermophilic ATP synthase (TF0F1)was sequenced and mutated. The advantage of using TF0F1 for mechanistic studies is its reconstitutability without MgATP. All genes for TF0F1 were arranged in the order of promotors, structural genes coding for the I, TF0 subunits (a, c, b) and TF1 subunits (δ, α, γ, β and e), and a terminator. The cause of the stability of these subunits was deduced from their sequence. The site-directed mutagenesis of the α and β subunits revealed that the 4 ionizable residues corresponding to Lys 21 and Asp 119 in the MgATP binding segments of adenylate kinase, are essential for the normal catalytic activity of this enzyme. The resulting βI164 and βN252 mutant subunits were both noncatalytic after reassembly into the αβγ subunit complex, even though both subunits bound significant amounts of ADP. The resulting αI175 reassembled weakly into an oligomer, while the αN261 was reconstituted into an αβγ subunit complex that showed no intersubunit cooperativity.