An intrinsic ATPase inhibitor binds near the active site of yeast mitochondrial F1-ATPase.
An ATPase inhibitor and its stabilizing factor, the 9K protein, are regulatory factors of F1F0-ATPase. The binding sites for these factors on F1 were examined using the zero length cross-linkers, N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline, and 1-ethyl-3-[3-dimethylamino)propyl]carbodiimide. The cross-linked products were analyzed by immunoblotting after SDS-polyacrylamide gel electrophoresis. The inhibitor and the 9K protein cross-linked to the alpha and beta subunits of F1, indicating that they interacted with both subunits. Peptide mapping and amino acid sequence analysis of the cross-linked products after weak acid hydrolysis showed that the inhibitor cross-linked to the Pro334-Asp363 region of the beta subunit. Amino acid sequence analysis of the cross-linked peptide showed that the inhibitor binds to Asp363 of the beta subunit. As this region contains the amino acid residues, including Tyr359, that are modified by nucleotide analogs and form the active site, the inhibitor probably binds to the catalytic site of F1.
Department of Food and Nutrition, Faculty of Human Life Science, Osaka City University, Sugimotochou.
N
N
Ichikawa
Y
Y
Yoshida
T
T
Hashimoto
K
K
Tagawa
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D001665:Binding Sites; D003432:Cross-Linking Reagents / Q000737:chemistry; D004791:Enzyme Inhibitors / Q000378:metabolism; D005022:Ethyldimethylaminopropyl Carbodiimide / Q000737:chemistry; D006868:Hydrolysis; D008928:Mitochondria / Q000201:enzymology*; D008969:Molecular Sequence Data; D010940:Plant Proteins / Q000378:metabolism; D006180:Proton-Translocating ATPases / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000378:metabolism*; D011804:Quinolines / Q000737:chemistry; D015003:Yeasts / Q000201:enzymology*
D000596:Amino Acids; D003432:Cross-Linking Reagents; D004791:Enzyme Inhibitors; C051630:HPS protein, soybean; D010940:Plant Proteins; D011804:Quinolines; C000470:EEDQ; D006180:Proton-Translocating ATPases; D005022:Ethyldimethylaminopropyl Carbodiimide