An Intrinsic ATPase Inhibitor Binds near the Active Site of Yeast Mitgchondrial F1-ATPase

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Abstract
An ATPase inhibitor and its stabilizing factor, the 9K protein, are regulatory factors of F1Fo-ATPase. The binding sites for these factors on F1 were examined using the zero length cross-linkers, N-ethoxycarbonyl-2-ethoxy-l,2-dihydroquinoline, and l-ethyl-3-[3-di-methylamino)propyl]carbodiimide. The cross-linked products were analyzed by immuno-blotting after SDS-polyacrylamide gel electrophoresis. The inhibitor and the 9K protein cross-linked to the α andβsubunits 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 βsubunit. Amino acid sequence analysis of the cross-linked peptide showed that the inhibitor binds to Asp363 of the βsubunit. 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

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