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 porti on (CF1) of the chloroplast ATP synthase by N-ethyhnaleimide was previ ously shown to inhibit ATP synthesis and hydrolysis. The gamma subunit interacts with the inhibitory epsilon subunit. Alkylation of gamma Cy s-89 could cause changes that result in the strengthening of the inter actions between the two subunits which would be inhibitory. We show th at the inhibition of the ATPase activity of CF1 in solution persists a fter 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 alkylat ed CF1 were similar, the rate of exchange of nucleotide bound to one s ite 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 conveni ently by stopped-flow fluorescence. The effects of N-ethylmaleimide mo dification 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 probabl y causes structural changes that perturb subunit and catalytic site in teractions.