Catalysis leads to posttranslational inactivation of the type 1 deiodinase and alters its conformation.

Previously, it was shown that the type 1 deiodinase (D1) is subject to substrate-dependent inactivation that is blocked by pretreatment with the inhibitor of D1 catalysis, propylthiouracil (PTU). Using HepG2 cells with endogenous D1 activity, we found that while considerable D1-mediated catalysis of reverse tri-iodothyronine (rT(3)) is observed in intact cells, there was a significant loss of D1 activity in sonicates assayed from the same cells in parallel. This rT(3)-mediated loss of D1 activity occurs despite no change in D1 mRNA levels and is blocked by PTU treatment, suggesting a requirement for catalysis. Endogenous D1 activity in sonicates was inactivated in a dose-dependent manner in HepG2 cells, with a ∼50% decrease after 10 nM rT(3) treatment. Inactivation of D1 was rapid, occurring after only half an hour of rT(3) treatment. D1 expressed in HEK293 cells was inactivated by rT(3) in a similar manner. (75)Se labeling of the D1 selenoprotein indicated that after 4 h rT(3)-mediated inactivation of D1 occurs without a corresponding decrease in D1 protein levels, though rT(3) treatment causes a loss of D1 protein after 8-24 h. Bioluminescence resonance energy transfer studies indicate that rT(3) exposure increases energy transfer between the D1 homodimer subunits, and this was lost when the active site of D1 was mutated to alanine, suggesting that a post-catalytic structural change in the D1 homodimer could cause enzyme inactivation. Thus, both D1 and type 2 deiodinase are subject to catalysis-induced loss of activity although their inactivation occurs via very different mechanisms.