PMID:8395501
| Citation |
Brandes, HK, Larimer, FW, Geck, MK, Stringer, CD, Schürmann, P and Hartman, FC (1993) Direct identification of the primary nucleophile of thioredoxin f. J. Biol. Chem. 268:18411-4 |
|---|---|
| Abstract |
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. |
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| Keywords |
Amino Acid Sequence; Base Sequence; Binding Sites; Chloroplasts/enzymology; Cysteine/chemistry; Cysteine/genetics; Enzyme Activation/drug effects; Escherichia coli/genetics; Fructose-Bisphosphatase/metabolism; Molecular Sequence Data; Mutagenesis, Site-Directed; Recombinant Fusion Proteins/chemistry; Recombinant Fusion Proteins/pharmacology; Thioredoxins/chemistry; Thioredoxins/genetics; Thioredoxins/pharmacology |
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