THE ROLE OF THE ACTIVE-SITE CYSTEINE IN CATALYSIS BY TYPE-1 IODOTHYRONINE DEIODINASE

Type 1 iodothyronine deiodinase (deiodinase 1) is a selenoenzyme that converts the prohormone T-4, to the active thyroid hormone T-3 by oute r ring deiodination or to the inactive metabolite rT(3), by inner ring deiodination. Although selenocysteine has been demonstrated to be ess ential for the biochemical profile of deiodinase 1, the role of a high ly conserved, active site cysteine (C124 in rat deiodinase 1) has not been defined. The present studies examined the effects of a Cys(124)Al a mutation on rat deiodinase 1 enzymatic function and substrate affini ty. At a constant 10-mM concentration of dithiothreitol (DTT), the C12 4A mutant demonstrated a a-fold lower apparent maximal velocity (V-max ) and K-m for rT(3) (K(m)rT(3)) than the wild type for outer ring deio dination, whereas the V-max/K-m ratio was unchanged. Similarly,the app arent V-max and KmT3 sulfate for inner ring deiodination were 2-fold l ower in the C124A mutant relative to those in the wild type, with no c hange in the V-max/K-m Patio. The C124A mutant exhibited ping-pong kin etics in the presence of DTT, and substitution of the active site cyst eine increased the KmDTT by 14-fold relative to that of the wild-type enzyme, with no significant effects on K(m)rT(3) or V-max. The C124A m utant was inhibited by propylthiouracil in an uncompetitive fashion an d exhibited a 2-fold increase in K,propylthiouracil compared with that of the wild type. K(m)rT(3), was also reduced for the C124A mutant wh en 5 mM reduced glutathione, a potential physiological monothiol cosub strate, was used in outer ring deiodination assays. These results demo nstrate that thiol cosubstrate interactions with C124 in type 1 deiodi nase play an important role in enhancing catalytic efficiency for both outer and inner ring deiodination.