TIGHT ATP AND ADP BINDING IN THE NONCATALYTIC SITES OF ESCHERICHIA-COLI F1-ATPASE IS NOT AFFECTED BY MUTATION OF BULKY RESIDUES IN THE GLYCINE-RICH LOOP

It is shown that ATP dissociates very slowly (koff< 6.4 × 105 s-1, t 1 2> 3 h) from the three noncatalytic sites of E. coli F1-ATPase and that ADP dissociates from these three sites in a homogeneous fashion with koff = 1.5 × 10-4 s-1 (t 1 2 = 1.35 h). Mutagenesis of α-subunit residues R171 and Q172 in the 'glycine-rich loop' (Homology A) consensus region of the noncatalytic sites was carried out to test the hypothesis that unusually bulky residues at these positions are responsible wholly or partly for the observed tight binding of adenine nucleotides. The mutations αQ172G or αR171S,Q172G had no effects on ATP or ADP binding to or rates of dissociation from F1 noncatalytic sites. Kd and Kd of isolated α-subunit were weakened by approximately 1 order of magnitude in both mutants. The results suggest that neither residue αR171 nor αQ172 interacts directly with bound nucleotide, and show that the presence of bulky residues per se in the glycine-rich loop region of F1-α-subunit is not responsible for tight binding in the noncatalytic sites. © 1990.