Formation and properties of hybrid photosynthetic F1-ATPases. Demonstration of different structural requirements for stimulation and inhibition by tentoxin.
A hybrid ATPase composed of cloned chloroplast ATP synthase beta and gamma subunits (betaC and gammaC) and the cloned alpha subunit from the Rhodospirillum rubrum ATP synthase (alphaR) was assembled using solubilized inclusion bodies and a simple single-step folding procedure. The catalytic properties of the assembled alpha3Rbeta3CgammaC were compared to those of the core alpha3Cbeta3CgammaC complex of the native chloroplast coupling factor 1 (CF1) and to another recently described hybrid enzyme containing R. rubrum alpha and beta subunits and the CF1 gamma subunit (alpha3Rbeta3RgammaC). All three enzymes were similarly stimulated by dithiothreitol and inhibited by copper chloride in response to reduction and oxidation, respectively, of the disulfide bond in the chloroplast gamma subunit. In addition, all three enzymes exhibited the same concentration dependence for inhibition by the CF1 epsilon subunit. Thus the CF1 gamma subunit conferred full redox regulation and normal epsilon binding to the two hybrid enzymes. Only the native CF1 alpha3Cbeta3CgammaC complex was inhibited by tentoxin, confirming the requirement for both CF1 alpha and beta subunits for tentoxin inhibition. However, the alpha3Rbeta3CgammaC complex, like the alpha3Cbeta3CgammaC complex, was stimulated by tentoxin at concentrations in excess of 10 microm. In addition, replacement of the aspartate at position 83 in betaC with leucine resulted in the loss of stimulation in the alpha3Rbeta3CgammaC hybrid. The results indicate that both inhibition and stimulation by tentoxin require a similar structural contribution from the beta subunit, but differ in their requirements for alpha subunit structure.
Tucker
W C
WC
Department of Molecular Biosciences, University of Kansas, Lawrence 66045, USA.
Du
Z
Z
Gromet-Elhanan
Z
Z
Richter
M L
ML
D017301:Ca(2+) Mg(2+)-ATPase / Q000378:metabolism; D000252:Calcium-Transporting ATPases / Q000378:metabolism; D002736:Chloroplasts / Q000201:enzymology; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D004926:Escherichia coli; D009183:Mycotoxins / Q000494:pharmacology*; D010084:Oxidation-Reduction; D010456:Peptides, Cyclic / Q000494:pharmacology*; D011487:Protein Conformation; D017510:Protein Folding; D006180:Proton-Translocating ATPases / Q000096:biosynthesis* / Q000378:metabolism; D013329:Structure-Activity Relationship
D004791:Enzyme Inhibitors; D009183:Mycotoxins; D010456:Peptides, Cyclic; D017301:Ca(2+) Mg(2+)-ATPase; D006180:Proton-Translocating ATPases; D000252:Calcium-Transporting ATPases; C003384:tentoxin