Characterizing substrate specificity and affinity in zebrafish deiodinases
Lee, Nicholas Yong Kyu
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Thyroid hormones are important for development and growth and their metabolism is mediated by a special class of enzymes called deiodinases. In this study, we cloned zebrafish deiodinases 1-3 (sequences from GenBank) and transfected them into mammalian cells. A special sequence called the selenocysteine insertion sequence was also cloned and transfected to express zebrafish deiodinases at high levels. Deiodination activity from the cloned zebrafish deiodinases indicated that GenBank sequences encode functional enzymes with the same specificity as human deiodinases. Zebrafish D1 was highly effective in catalyzing the outer ring deiodination of rT3. Zebrafish D2 catalyzed the outer ring deiodination of all tested substrates but showed no inner ring deiodination activity. Zebrafish D3 only catalyzed the inner ring deiodination of T3 into T2. We also observed that all zebrafish deiodinases required the SECIS element for enzyme activity. Furthermore, we demonstrated that the optimal temperature for zebrafish D3 catalyzed T3 deiodination is at room temperature instead of previously thought 28.5° C. The dramatic difference in zebrafish D3 (23.0° C compared to human D3 at 37.0° C) illustrated that there is an important difference between species. Finally, we demonstrated that zebrafish D3 has high affinity for T3 through Lineweaver Burk analysis and showed that the Km value of zebrafish D3 is in the low nanomolar range similar to human D3. Together with high substrate specificity for T3, we demonstrated that"zebrafish D3 is the primary inactiviator of T3 in zebrafish. We concluded that zebrafish deiodinase sequences in GenBank encode functional enzymes with high affinity and specificity but require the presence of the SECIS element for enzyme activity. Furthermore, we concluded that there is an important difference in optimal temperature between mammalian and zebrafish D3.
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