Catalytic Cooperativity in F₁-ATPase: Photoaffinity Labeling Studies with BzATP^a

Abstract

In 1982, Williams and Coleman (1982) introduced the photoaffinity label BzATP, (3’-o-[4-Benzoyl]Benzoyl Adenosine 5’-Triphosphate), in studies with the rat liver mitochondrial F₁-ATPase. A principal argument favoring the use of benzophenone-derivative photoaffinity labels is the very low probability of reaction between their excited state and water, the biochemical solvent. This situation contrasts dramatically with nitrene-generating photoprobes. The conclusions drawn from these initial studies emphasized that benzophenone-adenine nucleotide adducts could serve as informative photoactivatable substrate analogs for the nucleotide binding sites of this complex enzyme. With rat liver F₁, BzATP appeared to behave both as a fair substrate and also as a competitive inhibitor for ATPase activity. Under actinic illumination, the probe bound covalently to the F₁ in a site-specific manner, which was preventable by the presence of ATP. Furthermore, BzATP and BzABP were found, depending on experimental conditions, to seek out nucleotide-specific sites on the α and/or the β subunits of the enzyme. Upon light-activated, site-specific covalent binding to the rat liver F₁, ATPase activity was lost.