DIRECT IDENTIFICATION OF THE PRIMARY NUCLEOPHILE OF THIOREDOXIN-F

Thioredoxin, by virtue of the proximal active-site sulfhydryls (Trp-Cy s-Gly-Pro-Cys), catalyzes thiol-disulfide exchange with specific targe t enzymes. Considerable data (chemical modification, spectroscopic, an d crystallographic) have implicated the cysteinyl residue nearest the N terminus of thioredoxin as the primary nucleophile; however, direct proof has been lacking. Proof is now provided by characterization of s ite-directed mutants of thioredoxin f with respect to activation of ch loroplastic fructose-1,6-bisphosphatase (FBPase). The C49S mutant reta ins the capacity to activate FBPase, whereas the C46S mutant is totall y lacking in this regard. Based on kinetics of FBPase activation, wild -type and C49S thioredoxins exhibit half-saturation values of 0.9 and 4 muM, respectively. Lack of activation by C46S is not because of fail ure to interact with FBPase, for it exhibits a K(i) of 5 muM in compet ition with wild-type thioredoxin. Therefore, in the normal thioredoxin -catalyzed reduction pathway, Cys-46 is the nucleophile required to at tack the disulfide of the substrate and Cys-49 serves to cleave the mi xed disulfide intermediate, thus allowing for the release of oxidized thioredoxin and the reduced target enzyme.