Preparation and properties of a selenium-containing catalytic antibody as type I deiodinase mimic

Conversion of thyroxine (T-4) to 3,5,3'-triiodothyronine is an essential fi rst step in controlling thyroid hormone action. Type I deiodinase (DI) can catalyze the conversion to produce the bulk of serum 3,5,3'-triiodothyronin e. Acting as a mimic of DI, a selenium-containing catalytic antibody (Se-4C 5) prepared by converting the serine residues of monoclonal antibody 4C5 ra ised against T4 into selenocysteines, can catalyze the deiodination of T4 w ith dithiothreitol (DTT) as cosubstrate. The mimic enzyme Se-4C5 exhibited a much greater deiodinase activity than model compound ebselen and another selenium-containing antibody Se-Hp4 against GSH. The coupling of selenocyst eine with the combining pocket of antibody 4C5 endowed Se-4C5 with enzymati c activity. To probe the catalytic mechanism of the catalytic antibody, det ailed kinetic studies were carried out in this paper. Investigations into t he deiodinative reaction revealed the relationship between the initial velo city and substrate concentration. The characteristic parallel Dalziel plots demonstrated that Se-4C5-catalyzed reaction mechanism was ping-pong one, i nvolving at least one covalent enzyme intermediate. The kinetic properties of the catalytic antibody were similar to those of DI, with K-m values for T-4 and DTT of approximately 0.8 muM and 1.8 muM, respectively, and a V-m v alue of 270 pmol per mg of protein per min. The activity could be sensitive ly inhibited by 6-propyl-2-thiouracil (PTU) with a K-i value of similar to 120 muM at 2.0 muM T-4 concentration. The PTU inhibition was progressively alleviated with the increasing concentration of added DTT, revealing that P TU was a competitive inhibitor for DTT.