Directed mutagenesis of the dicyclohexylcarbodiimide-reactive carboxyl residues in β-subunit of F 1-ATPase of Escherichia coli

Abstract

Previous studies in which dicyclohexylcarbodiimide (DCCD) was used to inactivate F 1-ATPase enzymes have suggested that two glutamate residues in the β-subunit are essential for catalysis. In the Escherichia coli F 1-ATPase, these are residues β-Glu-181 and β-Glu-192. Oligonucleotide-directed mutagenesis was used to change these residues to β-Gln-181 and β-Gln-192. The β-Gln-181 mutation produced strong impairment of oxidative phosphorylation in vivo and also of ATPase and ATP-driven proton-pumping activities in membranes assayed in vitro. A low level of each activity was detected and an F 1-ATPase appeared to be assembled normally on the membranes. Therefore, it is suggested that the carboxyl side chain at residue β-181 is important, although not absolutely required, for catalysis in both directions on E. coli F 1-ATPase. The β-Gln-192 mutation produced partial inhibition of oxidative phosphorylation in vivo and membrane ATPase activity was reduced by 78%. These results contrast with the complete or near-complete inactivation seen when E. coli F 1-ATPase is reacted with DCCD and imply that DCCD-inactivation is attributable more to the attachment of the bulky DCCD molecule than to the derivatization of the carboxyl side chain of residue β-Glu-192. M. Ohtsubo and colleagues ( Biochem. Biophys. Res. Commun. (1987) 146, 705–710) described mutagenesis of the F 1-β-subunit of thermophilic bacterium PS3. Mutations (Glu → Gln) of the residues homologous to Glu-181 and Glu-192 of E. coli F 1-β-subunit both caused total inhibition of ATPase activity. Therefore, there was a marked difference in results obtained when the same residues were modified in the PS3 and E. coli F 1-β-Subunits.