| Thioltransferase (Glutaredoxin) is an 11,700 Da cytosolic protein that belongs to the family of thioldisulfide oxidoreductases (TDOR) (EC 1.8.4.1) that are involved in catalyzing thiol-disulfide exchange reactions. Along with other members of this family including thioredoxin and protein disulfide isomerase, thiol-transferase (TTase) is implicated in a variety of processes including cellular sulfhydryl homeostasis and protection of cellular enzymes from oxidative stress. It has previously been shown in our laboratory that TTase is specific for reduction of glutathionyl containing disulfides, implicating the glutathionyl disulfide of TTase (TTase-SSG) as an intermediate in the catalytic turnover. The focus of this study is (1) Determination of rate limiting step in TTase catalysis. (2) Selective inactivation of TTase.;In order to study the mechanism of action of TTase and to determine the rate limiting step in its reduction of glutathionyl containing disulfides, (35s) BSA-SSG was synthesized as the prototype substrate to study TTase catalysis by the radiolabel assay. The overall reaction of TTase-catalyzed reduction of BSA-SSG occurs in two steps: (1) TTase-S;Bronstead relationships for the non-enzymatic and enzymatic reactions revealed: (1) Both reactions follow linear free energy correlations. (2) The unusually low pKa of TTase-S;Since TTase contains nucleophilic cysteine at its active site, electrophilic agents like iodoacetate and N-ethyl maleimide can inactivate it. However these agents are non-specific and can inactivate any protein/enzyme that contains nucleophilic cysteines. In contrast, the fungal toxin, sporidesmin inactivated TTase selectively among the thiol disulfide oxidoreductase (TDOR) enzymes. Site directed mutagenesis of the different cysteines showed that C22, C25 and C82 are important for inactivation and form the basis of selective inactivation of TTase. Further sporidesmin inactivates TTase by forming a 1:1 covalent mixed disulfide complex in a process which requires molecular oxygen and is reversible by DTT only in the presence of urea. This indicates the formation of a mixed disulfide between sporidesmin and TTase that is shielded from reducing agent DTT. Other analogues of sporidesmin that contain the reactive dithiodioxopiperazine moiety and an aromatic benzyl group also inactivate TTase forming the basis of future studies on the minimal structural requirements for TTase inactivation. |
