Rapid Kinetics of Dehalogenation Promoted by Iodotyrosine Deiodinase from Human Thyroid

Abstract

Reductive dehalogenation such as that catalyzed by iodotyrosine deiodinase (IYD) is highly unusual in aerobic organisms but necessary for iodide salvage from iodotyrosine generated during thyroxine biosynthesis. Equally unusual is the dependence of this process on flavin. Rapid kinetics have now been used to define the basic processes involved in IYD catalysis. Time-dependent quenching of flavin fluorescence was used to monitor halotyrosine association to IYD. The substrates chloro-, bromo-, and iodotyrosine bound with similar rate constants (kon) ranging from 1.3 × 106 to 1.9 × 106 M–1 s–1. Only the inert substrate analogue fluorotyrosine exhibited a significantly (5-fold) slower kon (0.3 × 106 M–1 s–1). All data fit a standard two-state model and indicated that no intermediate complex accumulated during closure of the active site lid induced by substrate. Subsequent halide elimination does not appear to limit reactions of bromo- and iodotyrosine since both fully oxidized the reduced enzyme with nearly equivalent second-order rate constants (7.3 × 103 and 8.6 × 103 M–1 s–1, respectively) despite the differing strength of their carbon–halogen bonds. In contrast to these substrates, chlorotyrosine reacted with the reduced enzyme approximately 20-fold more slowly and revealed a spectral intermediate that formed at approximately the same rate as the bromo- and iodotyrosine reactions.

Active Site Binding Is Not Sufficient for Reductive Deiodination by Iodotyrosine Deiodinase

“…The steady-state parameter k cat / K m for turnover of I-Tyr by human IYD is also within two-fold of its second order k ox for I-Tyr dependent oxidation of FMN hq as measured by rapid kinetics. 8 Similarly, the k cat / K m value for I-Tyr is only 50% larger than that measured for I 2 -Tyr using a complementary 125 I-iodide release assay. 11 The lack of the zwitterion in 2IP greatly compromises its turnover by human IYD.…”

“…4,5 Iodide generation by IYD also provides a rare example of reductive dehalogenation in aerobic organisms and is even more unusual for its dependence on flavin mononucleotide (FMN) (Scheme 1). 6 This enzyme also catalyzes dehalogenation of chloro- and bromotyrosine 7,8 but appears limited to processing simple tyrosine derivatives. Thyroxine ( O -(4-hydroxy-3,5-diiodophenoxy)-3,5-diiodo-L-tyrosine) is also a tyrosine derivative but is not dehalogenated by IYD 9 and may not template a productive orientation of its active site residues.…”

A molecular view of the normal human thyroid structure and function reconstructed from its reference transcriptome map

“…They were followed by the known coding genes SLC26A7 , IYD , TSHR and SLC26A4 (solute carrier family 26 member 4) (Table 3 and Additional file 4). IYD catalyzes the deiodination of mono- and diiodotyrosine and is necessary for iodide salvage [37] and further production of hormones. The presence of abundant TSHRs on thyroid cells is crucial for their response to the main thyroid stimulating factor TSH.…”

The distribution and mechanism of iodotyrosine deiodinase defied expectations

“…One laboratory had access to radioactive [ 82 Br]-bromine and was first to report an IYD-dependent debromination of bromotyrosine (Br-Tyr) [8]. More recently, this activity was confirmed by the ability of Br-Tyr to discharge reducing equivalents from IYD containing reduced flavin (FMN hq ) and by detecting the product Tyr by reverse-phase (C-18) HPLC [32, 45, 53]. These same methods also revealed the ability of IYD to promote dechlorination of chlorotyrosine (Cl-Tyr) and its inability to promote defluorination of fluorotyrosine (F-Tyr).…”