The gamma subunit of the Escherichia coli F1-ATPase can be cross-linked near the glycine-rich loop region of a beta subunit when ADP + Mg2+ occupies catalytic sites but not when ATP + Mg2+ is bound.
A mutant of the Escherichia coli F1-ATPase, gamma S8C, has been reacted with a novel bifunctional reagent, N-maleimido-N'-(4-azido-2,3,5,6-tetrafluorobenzamido) cystamine (TFPAM-SS1). Modification of Cys-8 via the maleimide, followed by photolysis to convert the azido group to a reactive nitrene, led to cross-linking of the gamma subunit to a beta subunit. When this cross-linking was conducted with ADP + Mg2+ in catalytic sites, the predominant cross-linked product had a M(r) of 108,000. If cross-linking was done with uncleaved ATP + Mg2+ in catalytic sites, cross-linked products of 102,000 and 84,000 were formed. Cross-linking under both conditions led to inhibition of ATPase activity. TFPAM-SS1 could be cleaved by using reducing agents to break the disulfide bond that links the malemide and tetrafluorophenylazide moieties. Cleavage of this disulfide bond after formation of 102,000 and 84,000 species led to full recovery of ATPase activity. When the 108-kDa cross-linked product was cleaved, full activity was not restored, presumably because of insertion of the tetrafluorophenylazide into a functionally important site on the beta subunit. After cleavage of the disulfide bond, the free thiols could be reacted with [14C]N-ethylmaleimide, thereby radioactively tagging the sites of insertion of the tetrafluorophenylnitrene moiety. In this way, the site of cross-linking from Cys-8 of gamma to the beta subunit in the presence of ADP + Mg2+ was localized to within the sequence Val 145-Lys-155, which contains the glycine-rich loop. This loop region is a part of the catalytic site of the enzyme.
Aggeler
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R
Institute of Molecular Biology, University of Oregon, Eugene 97403.
Cai
S X
SX
Keana
J F
JF
Koike
T
T
Capaldi
R A
RA
D000244:Adenosine Diphosphate / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism*; D000595:Amino Acid Sequence; D001665:Binding Sites; D002384:Catalysis; D003432:Cross-Linking Reagents / Q000737:chemistry; D004926:Escherichia coli / Q000201:enzymology*; D005464:Fluorobenzenes / Q000737:chemistry; D005998:Glycine / Q000378:metabolism*; D008274:Magnesium / Q000378:metabolism*; D008301:Maleimides / Q000737:chemistry; D008969:Molecular Sequence Data; D006180:Proton-Translocating ATPases / Q000737:chemistry / Q000378:metabolism*
D003432:Cross-Linking Reagents; D005464:Fluorobenzenes; D008301:Maleimides; C073711:N-(4-azido-2,3,5,6-tetrafluorobenzyl)-3-maleimidopropionamide; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D006180:Proton-Translocating ATPases; D008274:Magnesium; D005998:Glycine