Alkylation of cysteine 89 of the gamma subunit of chloroplast coupling factor 1 with N-ethylmaleimide alters nucleotide interactions.
Alkylation of Cys-89 of the gamma subunit of the coupling factor portion (CF1) of the chloroplast ATP synthase by N-ethylmaleimide was previously shown to inhibit ATP synthesis and hydrolysis. The gamma subunit interacts with the inhibitory epsilon subunit. Alkylation of gamma Cys-89 could cause changes that result in the strengthening of the interactions between the two subunits which would be inhibitory. We show that the inhibition of the ATPase activity of CF1 in solution persists after removal of the epsilon subunit. Additionally, epsilon rebinds to control and alkylated CF1 at very similar CF1 to epsilon concentration ratios. Although the bound nucleotide contents of control and alkylated CF1 were similar, the rate of exchange of nucleotide bound to one site with medium nucleotide was accelerated by more than two orders of magnitude by alkylation. We show that ATP-induced release of bound 2'(3')-O-trinitrophenyl-adenosine 5'-diphosphate can be monitored conveniently by stopped-flow fluorescence. The effects of N-ethylmaleimide modification are likely to be felt over long distances. As determined by fluorescence resonance energy transfer, no nucleotide binding site so far mapped is closer than 5 nm to Cys-89. The bulky maleimide probably causes structural changes that perturb subunit and catalytic site interactions.
Soteropoulos
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Department of Biology, Johns Hopkins University, Baltimore, Maryland 21218.
Ong
A M
AM
McCarty
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D000227:Adenine Nucleotides / Q000737:chemistry*; D000244:Adenosine Diphosphate / Q000031:analogs & derivatives; D000266:Adenylyl Imidodiphosphate; D000478:Alkylation; D001665:Binding Sites; D002736:Chloroplasts / Q000737:chemistry*; D003545:Cysteine / Q000737:chemistry*; D005033:Ethylmaleimide / Q000737:chemistry*; D007425:Intracellular Membranes / Q000737:chemistry; D007700:Kinetics; D010084:Oxidation-Reduction; D010766:Phosphorylation; D010777:Photochemistry; D006180:Proton-Translocating ATPases / Q000737:chemistry*
D000227:Adenine Nucleotides; D000266:Adenylyl Imidodiphosphate; D000244:Adenosine Diphosphate; C036745:2',3'-(O-(2,4,6-trinitrocyclohexadienylidine))adenosine 5'-diphosphate; D006180:Proton-Translocating ATPases; D003545:Cysteine; D005033:Ethylmaleimide