Role of Asn-243 in the Phosphate-binding Subdomain of Catalytic Sites of Escherichia coli F1-ATPase*

In the catalytic mechanism of ATP synthase, phosphate (Pi) binding and release steps are believed to be correlated to -subunit rotation, and Pi binding is proposed to be prerequisite for binding ADP in the face of high cellular [ATP]/[ADP] ratios. In x-ray structures, residue Asn-243 appears centrally located in the Pibinding subdomain of catalytic sites. Here we studied the role of Asn-243 in Escherichia coli ATP synthase by mutagenesis to Ala and Asp. Mutation N243A caused 30-fold impairment of F1-ATPase activity; 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole inhibited this activity less potently than in wild type and Pi protected from inhibition. ADP-fluoroaluminate was more inhibitory than in wild-type, but ADP-fluoroscandium was less inhibitory. N243D F1-ATPase activity was impaired by 1300-fold and was not inhibited by ADP-fluoroaluminate or ADPfluoroscandium. 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole activated N243D F1-ATPase, and Pi did not affect activation. We conclude that residue Asn-243 is not involved in Pi binding directly but is necessary for correct organization of the transition state complex through extensive involvement in hydrogen bonding to neighboring residues. It is also probably involved in orientation of the “attacking water” and of an associated second water.