THE INTERPLAY OF SUBSTRATE, PROTEIN AND ITS COFACTOR IN CONTROLLING THE CATALYTIC PROPERTIES OF HUMAN IODOTYROSINE DEIODINASE
Rokita, Steven E
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Human iodotyrosine deiodinase (hIYD) belongs to nitro-FMN reductase superfamily and is responsible for recycling iodine from the I-Tyr (monoiodotyrosine) and I2-Tyr (diiodotyrosine) formed as byproducts of thyroid hormone synthesis. Heterologous expression of hIYD lacking its membrane domain in E. coli provided large quantities of highly pure hIYD that allowed for its physical and biochemical studies. Its kinetic parameters, binding constants and crystal structure were characterized. Substrate was able to induced dramatic effects on FMN coordination in hIYD, including the zwitterionic region of I-Tyr interacts with the N3 and O4 of the FMN, the OH of Thr239 moved close to N5 of FMN (from 4.8 Å to 3.1Å) and allowed the formation of a hydrogen bond. The aromatic ring of I-Tyr additionally stacks above the FMN. Accumulation of the neutral semiquinone was observed during the reduction of hIYD in the presence of substrate analogue 3-fluoro-L-tyrosine (F-Tyr). In the absence of ligand, only the oxidized and two-electron reduced forms of FMN were observed. Among all of the interactions to FMN in hIYD, H-bonding at the N5 FMN was identified as most important to control the redox of flavin. Mutagenesis of Thr239 to Ala removed this H-bonding as confirmed by the crystal structure of hIYDT239A*F-Tyr. As a result, no semiquinone was detected during the titration of hIYDT239A in the presence of F-Tyr. The deiodination activity of T239A was also dramatically decreased (10-fold). The zwitterion region of the substrate was found critical for binding to enzyme. Modifications of the zwitterion region resulted in at least a 500-fold increase in KD. The catalytic activity was unlikely determined by the zwitterion region since all of the modified substrates exhibited similar initial rates as the native substrate under conditions in which their concentration equaled their KD. The pH dependence of hIYD binding indicated that the phenolic group of I2-Tyr is also critical for binding and the hIYD prefers binding to the phenolate form of substrate. All the results presented in this thesis support the current proposed catalytic mechanism of IYD involving a stepwise electron transfer process.