Direct identification of the primary nucleophile of thioredoxin f.
Thioredoxin, by virtue of the proximal active-site sulfhydryls (Trp-Cys-Gly-Pro-Cys), catalyzes thiol-disulfide exchange with specific target enzymes. Considerable data (chemical modification, spectroscopic, and 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 site-directed mutants of thioredoxin f with respect to activation of chloroplastic fructose-1,6-bisphosphatase (FBPase). The C49S mutant retains the capacity to activate FBPase, whereas the C46S mutant is totally lacking in this regard. Based on kinetics of FBPase activation, wild-type and C49S thioredoxins exhibit half-saturation values of 0.9 and 4 microM, respectively. Lack of activation by C46S is not because of failure to interact with FBPase, for it exhibits a Ki of 5 microM in competition with wild-type thioredoxin. Therefore, in the normal thioredoxin-catalyzed reduction pathway, Cys-46 is the nucleophile required to attack the disulfide of the substrate and Cys-49 serves to cleave the mixed disulfide intermediate, thus allowing for the release of oxidized thioredoxin and the reduced target enzyme.
Protein Engineering Program, Oak Ridge-University of Tennessee Graduate School of Biomedical Science, Oak Ridge National Laboratory 37831.
H K
HK
Brandes
F W
FW
Larimer
M K
MK
Geck
C D
CD
Stringer
P
P
Schürmann
F C
FC
Hartman
D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D002736:Chloroplasts / Q000201:enzymology; D003545:Cysteine / Q000737:chemistry / Q000235:genetics; D004789:Enzyme Activation / Q000187:drug effects; D004926:Escherichia coli / Q000235:genetics; D006597:Fructose-Bisphosphatase / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000494:pharmacology; D013879:Thioredoxins / Q000737:chemistry* / Q000235:genetics / Q000494:pharmacology
D011993:Recombinant Fusion Proteins; D013879:Thioredoxins; D006597:Fructose-Bisphosphatase; D003545:Cysteine