PHOTOAFFINITY-LABELING OF THE ATP BINDING DOMAIN OF RUBISCO ACTIVASE AND A SEPARATE DOMAIN INVOLVED IN THE ACTIVATION OF RIBULOSE-1,5-BISPHOSPHATE CARBOXYLASE OXYGENASE/

Photoaffinity labeling of Rubisco activase with 2- and 8-N(3)ATP was u sed to identify the adenine binding domain for ATP. Rubisco activase h ydrolyzed both of these analogs of ATP and used their hydrolysis to su pport a low rate of Rubisco activation. When irradiated with ultraviol et light, these and other azido-substituted adenine nucleotides covale ntly modified Rubisco activase at two distinct binding sites. Competit ion binding experiments with ATP and ADP showed that one of the sites was the ATP binding domain. The other site was not a nucleotide bindin g domain per se but would bind adenine nucleotides if an azido moiety was present on the base. Tryptophan and other indoles prevented azidoa denine nucleotides from labeling this domain but afforded little prote ction to the ATP binding domain. The ability to selectively protect ea ch of the two binding sites made it possible to localize the adenine b inding domain for ATP to the region of Rubisco activase from N68-D74 a nd the other binding domain to a region near the N-terminus from Q10 t o D14. Modification of the region from Q10 to D14 by photoaffinity lab eling prevented Rubisco activase from promoting activation of Rubisco without affecting ATP hydrolysis. These data suggest that a specific r egion of Rubisco activase near the N-terminus may be a site of interac tion with Rubisco. Binding of azidoadenine nucleotides in this region appears to be fortuitous and may involve base-stacking with the specie s-invariant Trp at position 16 and hydrogen bonding of the azido moiet y.