Photoaffinity labeling of ribulose-1,5-bisphosphate carboxylase/ oxygenase activase with ATP γ-benzophenone: Identification of the ATP γ-phosphate binding domain

The phosphate-binding domain of the ATP-binding site of tobacco Rubisco (ribulose-1,5-bisphosphate carboxylase/oxygenase) activase was elucidated by photoaffinity labeling with a monoanhydride of ADP with N-(4-(benzoyl)phenylmethyl)phosphoramide ([γ-³²P] ATPγBP). Covalent incorporation of [γ-³²P]ATPγBP into the 42-kDa Rubisco activase subunit was dependent upon irradiation with ultraviolet light. Photolabeling of Rubisco activase with ATPγBP exhibited saturation kinetics; the apparent K_d for photolabeling was 5 μM. Two lines of evidence showed that ATPγBP modified Rubisco activase at the ATP-binding domain. First, physiological concentrations of ATP and ADP afforded complete protection against photolabeling of Rubisco activase by ATPγBP. Second, photolysis of Rubisco activase in the presence of ATPγBP decreased both the ATPase and the Rubisco activating activities. Inactivation of enzyme activity was dependent on ATPγBP concentration and could be prevented by including ADP during photolabeling. The region of Rubisco activase that was modified by ATPγBP was identified by isolating photolabeled peptides. Sequence analysis showed that ATPγBP modified Rubisco activase in two distinct regions; one region, S117-A136, is adjacent to the P-loop and the other region, V223-T234, exhibits homology to a region of adenylate kinase that ligates the essential metal ion. Photolabeling of these two regions of Rubisco activase was consistent with modification of the ATP γ-phosphate-binding domain of Rubisco activase with ATPγBP.