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-N3ATP was used to identify the adenine binding domain for ATP. Rubisco activase hydrolyzed both of these analogs of ATP and used their hydrolysis to support a low rate of Rubisco activation. When irradiated with ultraviolet light, these and other azido-substituted adenine nucleotides covalently modified Rubisco activase at two distinct binding sites. Competition binding experiments with ATP and ADP showed that one of the sites was the ATP binding domain. The other site was not a nucleotide binding domain per se but would bind adenine nucleotides if an azido moiety was present on the base. Tryptophan and other indoles prevented azidoadenine nucleotides from labeling this domain but afforded little protection to the ATP binding domain. The ability to selectively protect each of the two binding sites made it possible to localize the adenine binding domain for ATP to the region of Rubisco activase from N68-D74 and the other binding domain to a region near the N-terminus from Q10 to D14. Modification of the region from Q10 to D14 by photoaffinity labeling prevented Rubisco activase from promoting activation of Rubisco without affecting ATP hydrolysis. These data suggest that a specific region of Rubisco activase near the N-terminus may be a site of interaction with Rubisco. Binding of azidoadenine nucleotides in this region appears to be fortuitous and may involve base-stacking with the species-invariant Trp at position 16 and hydrogen bonding of the azido moiety.
Salvucci
M E
ME
U.S. Department of Agriculture, Agricultural Research Service, Lexington, Kentucky.
Chavan
A J
AJ
Klein
R R
RR
Rajagopolan
K
K
Haley
B E
BE
D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000378:metabolism* / Q000494:pharmacology; D000345:Affinity Labels / Q000378:metabolism / Q000494:pharmacology; D000595:Amino Acid Sequence; D001386:Azides / Q000378:metabolism / Q000494:pharmacology; D001665:Binding Sites; D004789:Enzyme Activation; D008969:Molecular Sequence Data; D010777:Photochemistry; D010940:Plant Proteins; D012273:Ribulose-Bisphosphate Carboxylase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D014364:Tryptophan / Q000031:analogs & derivatives / Q000494:pharmacology
D000345:Affinity Labels; D001386:Azides; D010940:Plant Proteins; C054765:rca protein, plant; C017040:8-azidoadenosine 5'-triphosphate; C042231:2-azidoadenosine 5'-triphosphate; D014364:Tryptophan; D000255:Adenosine Triphosphate; D012273:Ribulose-Bisphosphate Carboxylase