An attempt to convert noncatalytic nucleotide binding site of F1-ATPase to the catalytic site: hydrolysis of tethered ATP by mutated alpha subunits in the enzyme
Matsui, T.; Jault, J.M.; Allison, W.S.; Yoshida, M.
Biochemical and Biophysical Research Communications 220(1): 94-97
The alpha and beta subunits of F-1-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 beta subunits is occupied by a Gln residue in the noncatalytic nucleotide binding sites which reside in the alpha 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 a subunit with Glu-Arg and, reciprocally, the Glu in the catalytic site of the beta subunit with Gln. The resultant mutant alpha-3-beta-3-gamma complex lost steady-state ATPase activity. However, HPLC analysis of tryptic digests of the mutant alpha-3-beta-3-gamma complex which had been photolabeled with 2-N-3-(8-3H)ATP revealed that ATP tethered to the noncatalytic binding site was hydrolyzed, indicating that a primitive catalytic ability was generated at the alpha subunit by the introduced Glu.