Enzyme kinetic and substrate-binding studies of the thyroxine to 3,5,3'-triiodothyronine converting enzyme in the rat liver microsomal fraction.

The conversion of T4 to T3 was studied in the rat liver microsomal fraction. A mean Vmax of 0.11 pmol T3 produced per mg microsomal protein/min and a mean apparent Km of 2.1 muM T4 were found. An approximation to the real Km for the experimental conditions used was obtained by applying free instead of total T4 as the substrate concentration. Thus, the Km was found to be 9.7 nM free T4, and changes of Km with different amounts of microsomal protein added were not detected. rT3 was found to be a competitive inhibitor of the T4 to T3 conversion, with a mean apparent Ki of 9.4 nM rT3. Binding studies showed that T4 is bound not only nonspecifically but also to two different classes of specific binding sites. The dissociation constants were 7.5 and 1700 nM t4, and the maximal binding capacities were 58 and 4300 pmol T4/mg microsomal protein, respectively, rT3 and T3 both had one specific binding site besides their unspecific binding to the microsomal fraction. The dissociation constants were found to be 45 nM rT3 and 850 nM T3, respectively; the maximal binding capacities amounted to 75 pmol rT3 and 4600 pmol T3 per mg microsomal protein, respectively. rT3 competes with T4 for its first (apparent Ki, 55 nM rT3) and T3 competes with T4 for its second specific binding site (apparent Ki, 960 nM T3). It is suggested, that the first specific binding site for T4 and the specific binding site for rT3 are identical, and that they represent the 5'-deiodinase. The rT3-induced inhibition of the T4 to T3 conversion is caused by a competition for the binding site of the enzyme. A competition for cofactors may play an additional role. T3 competes with T4 for a different specific binding site, which may contain the 5-deiodinase.