Preparation and properties of a selenium-containing catalytic antibody as type I deiodinase mimic.
Conversion of thyroxine (T4) to 3,5,3'-triiodothyronine is an essential first step in controlling thyroid hormone action. Type I deiodinase (DI) can catalyze the conversion to produce the bulk of serum 3,5,3'-triiodothyronine. Acting as a mimic of DI, a selenium-containing catalytic antibody (Se-4C5) prepared by converting the serine residues of monoclonal antibody 4C5 raised against T4 into selenocysteines, can catalyze the deiodination of T4 with 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 selenocysteine with the combining pocket of antibody 4C5 endowed Se-4C5 with enzymatic activity. To probe the catalytic mechanism of the catalytic antibody, detailed kinetic studies were carried out in this paper. Investigations into the deiodinative reaction revealed the relationship between the initial velocity and substrate concentration. The characteristic parallel Dalziel plots demonstrated that Se-4C5-catalyzed reaction mechanism was ping-pong one, involving at least one covalent enzyme intermediate. The kinetic properties of the catalytic antibody were similar to those of DI, with Km values for T4 and DTT of approximately 0.8 microm and 1.8 mm, respectively, and a Vm value of 270 pmol per mg of protein per min. The activity could be sensitively inhibited by 6-propyl-2-thiouracil (PTU) with a K(i) value of approximately 120 microm at 2.0 microm T4 concentration. The PTU inhibition was progressively alleviated with the increasing concentration of added DTT, revealing that PTU was a competitive inhibitor for DTT.
Lian
G
G
Laboratory of Rare Earth Chemistry and Physics, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun 130022, People's Republic of China.
Ding
L
L
Chen
M
M
Liu
Z
Z
Zhao
D
D
Ni
J
J
D000818:Animals; D000906:Antibodies / Q000737:chemistry*; D000911:Antibodies, Monoclonal / Q000737:chemistry; D001667:Binding, Competitive; D002384:Catalysis; D020134:Catalytic Domain; D004229:Dithiothreitol / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D006241:Haptens / Q000737:chemistry; D006801:Humans; D006825:Hybridomas / Q000378:metabolism; D007453:Iodide Peroxidase / Q000737:chemistry*; D007700:Kinetics; D051379:Mice; D008807:Mice, Inbred BALB C; D008956:Models, Chemical; D011441:Propylthiouracil / Q000494:pharmacology; D011863:Radioimmunoassay; D012643:Selenium / Q000737:chemistry*; D014407:Tumor Cells, Cultured
D000906:Antibodies; D000911:Antibodies, Monoclonal; D006241:Haptens; D011441:Propylthiouracil; D007453:Iodide Peroxidase; D012643:Selenium; D004229:Dithiothreitol