Alkylation of Cys-89 of the γ 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 γ subunit interacts with the inhibitory ε subunit. Alkylation of γ 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 ε subunit. Additionally, ε rebinds to control and alkylated CF1 at very similar CF1 to ε 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.
|Original language||English (US)|
|Number of pages||7|
|Journal||Journal of Biological Chemistry|
|State||Published - Aug 5 1994|
All Science Journal Classification (ASJC) codes
- Molecular Biology
- Cell Biology