An Attempt to Convert Noncatalytic Nucleotide Binding Site of F1-ATPase to the Catalytic Site: Hydrolysis of Tethered ATP by Mutated α Subunits in the Enzyme

Abstract

The α and β subunits of F1-ATPase are homologous in primary structure and have similar folding topologies. The position of the essential Glu residue in the catalytic sites which reside in the β subunits is occupied by a Gln residue in the noncatalytic nucleotide binding sites which reside in the α subunits. To test if an exchange of catalytic and noncatalytic binding sites is possible, we have replaced the Gln-Lys sequence in the noncatalytic binding site of the α subunit with Glu-Arg and, reciprocally, the Glu in the catalytic site of the β subunit with Gln. The resultant mutant α3β3γ complex lost steady-state ATPase activity. However, HPLC analysis of tryptic digests of the mutant α3β3γ complex which had been photolabeled with 2-N3-[8-3H]ATP revealed that ATP tethered to the noncatalytic binding site was hydrolyzed, indicating that a primitive catalytic ability was generated at the α subunit by the introduced Glu.