F1-ATPase with cysteine instead of serine at residue 373 of the alpha subunit.
Escherichia coli strain AN718 contains the alpha S373F mutation in F1F0-ATP synthase which blocks ATP synthesis (oxidative phosphorylation) and steady-state F1-ATPase activity. The revertant strain AN718SS2 containing the mutation alpha C373 was isolated and shown to confer a phenotype of higher growth yield than that of the wild type in liquid medium containing limiting glucose, succinate, or LB. Purified F1 from strain AN718SS2 was found to have 30% of wild-type steady-state ATPase activity and 60% of wild-type oxidative phosphorylation activity. Azide sensitivity of ATPase activity and ADP-induced enhancement of bound aurovertin fluorescence, both of which are lost in alpha S373F mutant F1, were regained in alpha C373 F1. N-Ethylmaleimide (NEM) inactivated alpha C373 F1 steady-state ATPase potently but had no effect on unisite ATPase. Complete inactivation of alpha C373 F1 steady-state ATPase corresponded to incorporation of one NEM per F1 (mol/mol), in just one of the three alpha subunits. NEM-inactivated enzyme showed azide-insensitive residual ATPase activity and loss of ADP-induced enhancement of bound aurovertin fluorescence. The data confirm the view that placement at residue alpha 373 of a bulky amino acid side-chain (phenylalanyl or NEM-derivatized cysteinyl) blocks positive catalytic cooperativity in F1. The fact that NEM inhibits steady-state ATPase when only one alpha subunit of three is reacted suggests a cyclical catalytic mechanism.
Lee
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RS
Department of Biochemistry, University of Rochester Medical Center, New York 14642.
Wilke-Mounts
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S
Senior
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AE
D000595:Amino Acid Sequence; D002462:Cell Membrane / Q000201:enzymology; D003545:Cysteine; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development; D005033:Ethylmaleimide / Q000494:pharmacology; D005838:Genotype; D007700:Kinetics; D046911:Macromolecular Substances; D006180:Proton-Translocating ATPases / Q000235:genetics* / Q000378:metabolism; D012694:Serine; D013045:Species Specificity
D046911:Macromolecular Substances; D012694:Serine; D006180:Proton-Translocating ATPases; D003545:Cysteine; D005033:Ethylmaleimide