Substitution of cysteine for a conserved alanine residue in the catalytic center of type II iodothyronine deiodinase alters interaction with reducing cofactor.

UNLABELLED Human type II iodothyronine deiodinase (D2) catalyzes the activation of T(4) to T(3). The D2 enzyme, like the type I (D1) and type III (D3) deiodinases, contains a selenocysteine (SeC) residue (residue 133 in D2) in the highly conserved catalytic center. Remarkably, all of the D2 proteins cloned so far have an alanine two residue-amino terminal to the SeC, whereas all D1 and D3 proteins contain a cysteine at this position. A cysteine residue in the catalytic center could assist in enzymatic action by providing a nucleophilic sulfide or by participating in redox reactions with a cofactor or enzyme residues. We have investigated whether D2 mutants with a cysteine (A131C) or serine (A131S) two-residue amino terminal to the SeC are enzymatically active and have characterized these mutants with regard to substrate affinity, reducing cofactor interaction and inhibitor profile. COS cells were transfected with expression vectors encoding wild-type (wt) D2, D2 A131C, or D2 A131S proteins. Kinetic analysis was performed on homogenates with dithiothreitol (DTT) as reducing cofactor. The D2 A131C and A131S mutants displayed similar Michaelis-Menten constant values for T(4) (5 nM) and reverse T(3) (9 nM) as the wt D2 enzyme. The limiting Michaelis-Menten constant for DTT of the D2 A131C enzyme was 3-fold lower than that of the wt D2 enzyme. The wt and mutant D2 enzymes are essentially insensitive to propylthiouracil [concentration inhibiting 50% of activity (IC(50)) > 2 mM] in the presence of 20 mM DTT, but when tested in the presence of 0.2 mM DTT the IC(50) value for propylthiouracil is reduced to about 0.1 mM. During incubations of intact COS cells expressing wt D2, D2 A131C, or D2 A131S, addition of increasing amounts of unlabeled T(4) resulted in the saturation of [(125)I]T(4) deiodination, as reflected in a decrease of [(125)I]T(3) release into the medium. Saturation first appeared at medium T(4) concentrations between 1 and 10 nM. IN CONCLUSION substitution of cysteine for a conserved alanine residue in the catalytic center of the D2 protein does not inactivate the enzyme in vitro and in situ, but rather improves the interaction with the reducing cofactor DTT in vitro.