Rotation of nucleotide sites is not required for the enzymic activity of chloroplast coupling factor 1

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Abstract
New heterobifunctional photoaffinity cross-linking reagents, 6-maleimido-N-(4-benzoylphenyl)hexanamide, 12-maleimido-N-(4-benzoylphenyl)dodecanamide, and 12-[14C]maleimido-N-(4-benzoylphenyl)dodecanamide, were synthesized to investigate the mechanism of ATP hydrolysis by chloroplast coupling factor 1. These reagents react with sulfhydryl groups on the .gamma.-polypeptide. Subsequent photolysis cross-links the .gamma.-polypeptide covalently to .alpha.- and .beta.-polypeptides. The cross-linkers prevent major movements of the .gamma.-polypeptide with respect to the .alpha.- and .beta.-polypeptides but are sufficiently long to permit some flexibility in the enzyme structure. When .apprx. 50% of the .gamma.-polypeptide was cross-linked to .alpha.- and .beta.-polypeptides, a 7% loss in ATPase activity was observed for the longer cross-linker and a 12% loss for the shorter. These results indicate that large movements of .alpha.- and .beta.-polypeptides with respect to the .gamma.-polypeptide are not essential for catalysis. In particular, rotation of the polypeptide chains to create structurally equivalent sites during catalysis is not a required feature of the enzyme mehanism.