IDENTIFICATION OF CRITICAL AMINO-ACIDS FOR 3,5,3'-TRIIODOTHYRONINE DEIODINATION BY HUMAN TYPE-1 DEIODINASE BASED ON COMPARATIVE FUNCTIONAL-STRUCTURAL ANALYSES OF THE HUMAN, DOG, AND RAT ENZYMES

The selenoenzyme, type 1 iodothyronine deiodinase (type 1 DI), catalyz es the activation of thyroxine (T4) to 3,5,3'-triiodothyronine (T3) bu t 3,3',5'-triiodothyronine (rT3) is the preferred substrate for the hu man enzyme. Since the dog type 1 DI has a significantly lower affinity for rT3, we cloned the dog type 1 DI to identify amino acids critical for rT3 binding. The K-m of the transiently expressed dog enzyme for rT3 5'-deiodination is 25-fold higher than that of the human enzyme. H owever, the K-i of T4 for rT3 deiodination by dog type 1 DI is only 3- fold higher than that for the human, suggesting that the differences b etween the two proteins affect binding of rT3 more than that of T4. Co mparative competition studies in which rT3 or T4 is used to block cova lent bromoacetyl T3 binding to the two proteins support this. Mutation al studies showed that the critical differences between the dog (D) an d human (H) enzymes are Asn (D) 45 Gly (H), Gly (D) 46 Glu (H), and Le u (D) 60:Phe (H) 65. Substitution of the human residues for those of t he dog at these positions causes Che predicted changes in the K-m (rT3 ) and vice versa. A Phe(65) to Leu mutation alone in the human enzyme increases the K-m (rT3) 10-fold. We speculate that phe(65) is especial ly important for rT3 binding due to an interaction between the tyrosyl ring of rT3 and the aromatic ring of Phe(65).