In vitro assembly of the core catalytic complex of the chloroplast ATP synthase.
The regulatory gamma subunit and an alpha beta complex were isolated from the catalytic F1 portion of the chloroplast ATP synthase. The isolated gamma subunit was devoid of catalytic activity, whereas the alpha beta complex exhibited a very low ATPase activity (approximately 200 nmol/min/mg of protein). The alpha beta complex migrated as a hexameric alpha 3 beta 3 complex during ultracentrifugation and gel filtration but reversibly dissociated into alpha and beta monomers after freezing and thawing in the presence of ethylenediamine tetraacetic acid and in the absence of nucleotides. Conditions are described in which the gamma and alpha beta preparations were combined to rapidly and efficiently reconstitute a fully functional catalytic core enzyme complex. The reconstituted enzyme exhibited normal tight binding and sensitivity to the inhibitory epsilon subunit and to the allosteric inhibitor tentoxin. However, neither the alpha beta complex nor the isolated gamma subunit alone could bind the epsilon subunit or tentoxin with high affinity. Similarly, high affinity binding sites for ATP and ADP, which are characteristic of the core alpha 3 beta 3 gamma enzyme, were absent from the alpha beta complex. The results indicate that when the gamma subunit binds to the alpha beta complex, it induces a three-dimensional conformation in the enzyme, which is necessary for tight binding of the inhibitors and for high-affinity, asymmetric nucleotide binding.
Gao
F
F
Department of Biochemistry, University of Kansas, Lawrence 66045, USA.
Lipscomb
B
B
Wu
I
I
Richter
M L
ML
D000244:Adenosine Diphosphate / Q000378:metabolism; D001665:Binding Sites; D002384:Catalysis; D002736:Chloroplasts / Q000201:enzymology*; D002850:Chromatography, Gel; D004789:Enzyme Activation; D008970:Molecular Weight; D009183:Mycotoxins / Q000378:metabolism; D010456:Peptides, Cyclic / Q000378:metabolism; D006180:Proton-Translocating ATPases / Q000302:isolation & purification / Q000378:metabolism*
D009183:Mycotoxins; D010456:Peptides, Cyclic; D000244:Adenosine Diphosphate; D006180:Proton-Translocating ATPases; C003384:tentoxin