Kinetic and mutational analyses of the regulation of phosphoribulokinase by thioredoxins

Despite little supportive data, differential target protein susceptibility to redox regulation by thioredoxin (Trx) fend Trx in has been invoked to ac count for two distinct Trxs in chloroplasts. However, this postulate has no t been rigorously tested with phosphoribulokinase (PRK), a fulcrum for redo x regulation of the Calvin cycle. Prerequisite to Trx studies, the activati on of spinach PRK by dithiothreitol, 2-mercaptoethanol, and glutathione was examined. Contrary to prior reports, each activated PRK, but only dithioth reitol supported Trx-dependent activation. Comparative kinetics of activati on of PRK showed Trx m to be more efficient than Trx f because of its 40% h igher V-max but similar S-0.5. Activations were insensitive to ribulosebisp hosphate carboxylase, which may complex with PRK in vivo. To probe the basi s for superiority of Trx m, we characterized site-directed mutants of Trx f , in which unique residues in conserved regions were replaced with Trx m co unterparts or deleted. These changes generally resulted in V-max enhancemen ts, the largest (6-fold) of which occurred with T105I, reflective of substi tution in a hydrophobic region that opposes the active site. inclusive of t he present study activation kinetics of several different Trx-regulated enz ymes indicate redundancy in the functions of the chloroplastic Trxs.