Role of betaAsn-243 in the phosphate-binding subdomain of catalytic sites of Escherichia coli F(1)-ATPase.
In the catalytic mechanism of ATP synthase, phosphate (P(i)) binding and release steps are believed to be correlated to gamma-subunit rotation, and P(i) binding is proposed to be prerequisite for binding ADP in the face of high cellular [ATP]/[ADP] ratios. In x-ray structures, residue betaAsn-243 appears centrally located in the P(i)-binding subdomain of catalytic sites. Here we studied the role of betaAsn-243 in Escherichia coli ATP synthase by mutagenesis to Ala and Asp. Mutation betaN243A caused 30-fold impairment of F(1)-ATPase activity; 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole inhibited this activity less potently than in wild type and P(i) protected from inhibition. ADP-fluoroaluminate was more inhibitory than in wild-type, but ADP-fluoroscandium was less inhibitory. betaN243D F(1)-ATPase activity was impaired by 1300-fold and was not inhibited by ADP-fluoroaluminate or ADP-fluoroscandium. 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole activated betaN243D F(1)-ATPase, and P(i) did not affect activation. We conclude that residue betaAsn-243 is not involved in P(i) 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.
Department of Biochemistry and Biophysics, University of Rochester Medical Center, Rochester, New York 14642, USA.
Zulfiqar
Z
Ahmad
Alan E
AE
Senior
D000244:Adenosine Diphosphate / Q000494:pharmacology; D001665:Binding Sites; D020134:Catalytic Domain; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins / Q000737:chemistry*; D006860:Hydrogen Bonding; D010069:Oxadiazoles / Q000494:pharmacology; D010710:Phosphates / Q000378:metabolism*; D006180:Proton-Translocating ATPases / Q000037:antagonists & inhibitors / Q000737:chemistry*
D029968:Escherichia coli Proteins; D010069:Oxadiazoles; D010710:Phosphates; D000244:Adenosine Diphosphate; C083020:7-chloro-4-sulfobenzoxadiazole; D006180:Proton-Translocating ATPases