Spinach chloroplast coupling factor CF1-alpha 3 beta 3 core complex: structure, stability, and catalytic properties.
A minimal chloroplast coupling factor CF1 core complex, containing only alpha and beta subunits, has been isolated from spinach thylakoids [Avital, S., & Gromet-Elhanan, Z. (1991) J. Biol. Chem. 266, 7067-7072]. This CF1(alpha beta) exhibited a low MgATPase activity, which was stimulated but not inhibited by low concentrations of the species-specific CF1 effector tentoxin. As is reported here, the structure of CF1(alpha beta) could not be determined due to its instability. However, its pretreatment with high tentoxin concentrations resulted in a remarkable 50-fold stimulation of the MgATPase activity as well as stabilization of its hexameric structure, thus enabling the isolation of a more active CF1-alpha 3 beta 3 complex by size-exclusion chromatography. A detailed characterization of the MgATPase activity of this tentoxin-stabilized CF1-alpha 3 beta 3 hexamer, as compared to the activity of a CF1 complex lacking the epsilon subunit, revealed similar apparent Km values and a similar stimulation by the presence of 100 microM tentoxin in the assay medium, but drastic differences in all other tested assays. Most pronounced were their different temperature profiles and different responses to all added inhibitors and stimulators of the CF1 MgATPase activity and to excess free Mg2+ ions. The specific properties of the stable CF1-alpha 3 beta 3 hexamer are identical to those earlier reported for its parent-unstable CF1(alpha beta). These results indicate that, although the CF1 gamma subunit is not required for the low CF1(alpha beta) ATPase activity nor for the higher activity of the tentoxin-stabilized CF1-alpha 3 beta 3, it plays a central role in obtaining the typical functional properties of the CF1-ATPase. Kinetic cooperativity could not be critically tested as yet with any F1-alpha 3 beta 3. However, tentoxin, as azide, has been shown to inhibit multisite but not unisite catalysis. Therefore, the observation that CF1-alpha 3 beta 3 is only stimulated by tentoxin suggests that the required presence of CF1-gamma for obtaining inhibition by tentoxin reflects the role of this subunit in cooperative interactions between the catalytic sites.
Sokolov
M
M
Department of Biochemistry, Weizmann Institute of Science, Rehovot, Israel.
Gromet-Elhanan
Z
Z
D000255:Adenosine Triphosphate / Q000378:metabolism; D017301:Ca(2+) Mg(2+)-ATPase / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism; D002736:Chloroplasts / Q000201:enzymology*; D002851:Chromatography, High Pressure Liquid; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors; D004795:Enzyme Stability; D006868:Hydrolysis; D046911:Macromolecular Substances; D010456:Peptides, Cyclic / Q000494:pharmacology; D011487:Protein Conformation; D006180:Proton-Translocating ATPases / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism*; D018724:Spinacia oleracea / Q000201:enzymology
D004791:Enzyme Inhibitors; D046911:Macromolecular Substances; D010456:Peptides, Cyclic; D000255:Adenosine Triphosphate; D017301:Ca(2+) Mg(2+)-ATPase; D006180:Proton-Translocating ATPases; C003384:tentoxin