Inverse regulation of F1-ATPase activity by a mutation at the regulatory region on the gamma subunit of chloroplast ATP synthase.
Chloroplast ATP synthase is a thiol-modulated enzyme whose DeltamuH(+)-linked activation is strongly influenced by reduction and the formation of a disulphide bridge between Cys(199) and Cys(205) on the gamma subunit. In solubilized chloroplast coupling factor 1 (CF(1)), reduction of the disulphide bond elicits the latent ATP-hydrolysing activity. To assess the regulatory importance of the amino acid residues around these cysteine residues, we focused on the three negatively charged residues Glu(210)-Asp-Glu(212) close to the two cysteine residues and also on the following region from Leu(213) to Ile(230), and investigated the modulation of ATPase activity by chloroplast thioredoxins. The mutant gamma subunits were reconstituted with the alpha and beta subunits from F(1) of the thermophilic bacterium Bacillus PS3; the active ATPase complexes obtained were purified by gel-filtration chromatography. The complex formed with a mutant gamma subunit in which Glu(210) to Glu(212) had been deleted was inactivated rather than activated by reduction of the disulphide bridge by reduced thioredoxin, indicating inverse regulation. This complex was insensitive to the inhibitory CF(1)-epsilon subunit when the mutant gamma subunit was oxidized. In contrast, the deletion of Glu(212) to Ile(230) converted the complex from a modulated state into a highly active state.
Konno
H
H
Chemical Resources Laboratory, Tokyo Institute of Technology, Nagatsuta 4259, Midori-Ku, Yokohama 226-8503, Japan.
Yodogawa
M
M
Stumpp
M T
MT
Kroth
P
P
Strotmann
H
H
Motohashi
K
K
Amano
T
T
Hisabori
T
T
D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000235:genetics; D001407:Bacillus / Q000201:enzymology; D002736:Chloroplasts / Q000201:enzymology*; D004220:Disulfides / Q000378:metabolism; D004229:Dithiothreitol / Q000378:metabolism / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics*; D010084:Oxidation-Reduction / Q000187:drug effects; D010940:Plant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D021122:Protein Subunits; D006180:Proton-Translocating ATPases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D019163:Reducing Agents / Q000378:metabolism / Q000494:pharmacology; D012399:Rotation; D013879:Thioredoxins / Q000378:metabolism / Q000494:pharmacology
D004220:Disulfides; D010940:Plant Proteins; D021122:Protein Subunits; D011993:Recombinant Fusion Proteins; D019163:Reducing Agents; D013879:Thioredoxins; D000255:Adenosine Triphosphate; D006180:Proton-Translocating ATPases; D004229:Dithiothreitol