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Directed mutagenesis of the dicyclohexylcarbodiimide-reactive carboxyl residues in beta-subunit of F1-ATPase of Escherichia coli.

Abstract：

:Previous studies in which dicyclohexylcarbodiimide (DCCD) was used to inactivate F1-ATPase enzymes have suggested that two glutamate residues in the beta-subunit are essential for catalysis. In the Escherichia coli F1-ATPase, these are residues beta-Glu-181 and beta-Glu-192. Oligonucleotide-directed mutagenesis was used to change these residues to beta-Gln-181 and beta-Gln-192. The beta-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 F1-ATPase appeared to be assembled normally on the membranes. Therefore, it is suggested that the carboxyl side chain at residue beta-181 is important, although not absolutely required, for catalysis in both directions on E. coli F1-ATPase. The beta-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 F1-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 beta-Glu-192. M. Ohtsubo and colleagues (Biochem. Biophys. Res. Commun. (1987) 146, 705-710) described mutagenesis of the F1-beta-subunit of thermophilic bacterium PS3. Mutations (Glu----Gln) of the residues homologous to Glu-181 and Glu-192 of E. coli F1-beta-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 F1-beta-subunits.

journal_name

Arch Biochem Biophys

journal_title

Archives of biochemistry and biophysics

authors

Parsonage D,Wilke-Mounts S,Senior AE

doi

10.1016/0003-9861(88)90121-x

subject

Has Abstract

pub_date

1988-02-15 00:00:00

pages

222-5

issue

eissn

0003-9861

issn

1096-0384

pii

0003-9861(88)90121-X

journal_volume

261

pub_type

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Directed mutagenesis of the dicyclohexylcarbodiimide-reactive carboxyl residues in beta-subunit of F1-ATPase of Escherichia coli.