N-ethylmaleimide-sensitive mutant (beta Val-153-->Cys) Escherichia coli F1-ATPase: cross-linking of the mutant beta subunit with the alpha subunit.
A beta subunit mutation, beta Val-153-->Cys, in the glycine-rich sequence (phosphate-binding loop) of Escherichia coli F1 was constructed. Like vacuolar-type ATPase, the mutant enzyme was inhibited by N-ethylmaleimide (NEM) and labeled with [14C]NEM. The inhibition and labeling were prevented by ATP. m-Maleimidobenzoyl-N-hydroxysuccinimide (MBS) (3 microM) almost completely inhibited the mutant enzyme, and cross-linked one pair of alpha and beta subunits. These results suggest that the interaction of the domain near beta Val-153 with the alpha subunit is essential for catalytic cooperativity of the enzyme and that beta Val-153 is within 10 A of the alpha subunit.
Iwamoto
A
A
Department of Organic Chemistry and Biochemistry, Osaka University, Japan.
Orita-Saita
Y
Y
Maeda
M
M
Futai
M
M
D000255:Adenosine Triphosphate / Q000494:pharmacology; D000595:Amino Acid Sequence; D003432:Cross-Linking Reagents / Q000494:pharmacology; D003545:Cysteine / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology*; D005033:Ethylmaleimide / Q000494:pharmacology*; D008969:Molecular Sequence Data; D009154:Mutation / Q000502:physiology; D006180:Proton-Translocating ATPases / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics; D013388:Succinimides / Q000494:pharmacology
D003432:Cross-Linking Reagents; D013388:Succinimides; D000255:Adenosine Triphosphate; D006180:Proton-Translocating ATPases; D003545:Cysteine; D005033:Ethylmaleimide