PHOTOAFFINITY-LABELING OF RIBULOSE-1,5-BISPHOSPHATE CARBOXYLASE OXYGENASE ACTIVASE WITH ATP GAMMA-BENZOPHENONE - IDENTIFICATION OF THE ATP GAMMA-PHOSPHATE BINDING DOMAIN

The phosphate-binding domain of the ATP-binding site of tobacco Rubisc o (ribulose-1,5-bisphosphate carboxylase/oxygenase) activase was eluci dated by photoaffinity labeling with a monoanhydride of ADP with N-(4- (benzoyl)phenylmethyl)phosphoramide ([gamma-P-32] ATPgammaBP). Covalen t incorporation of [gamma-P-32]ATPgammaBP into the 42-kDa Rubisco acti vase subunit was dependent upon irradiation with ultraviolet light. Ph otolabeling of Rubisco activase with ATPgammaBP exhibited saturation k inetics; the apparent K(d) for photolabeling was 5 muM. Two lines of e vidence showed that ATPgammaBP modified Rubisco activase at the ATP-bi nding domain. First, physiological concentrations of ATP and ADP affor ded complete protection against photolabeling of Rubisco activase by A TPgammaBP. Second, photolysis of Rubisco activase in the presence of A TPgammaBP decreased both the ATPase and the Rubisco activating activit ies. Inactivation of enzyme activity was dependent on ATPgammaBP conce ntration and could be prevented by including ADP during photolabeling. The region of Rubisco activase that was modified by ATPgammaBP was id entified by isolating photolabeled peptides. Sequence analysis showed that ATPgammaBP modified Rubisco activase in two distinct regions; one region, S117-A136, is adjacent to the P-loop and the other region, V2 23-T234, exhibits homology to a region of adenylate kinase that ligate s the essential metal ion. Photolabeling of these two regions of Rubis co activase was consistent with modification of the ATP gamma-phosphat e-binding domain of Rubisco activase with ATPgammaBP.