Abstract
Active site lids are common features of enzymes and typically undergo conformational changes upon substrate binding to promote catalysis. Iodotyrosine deiodinase is no exception and contains a lid segment in all of its homologues from human to bacteria. The solution-state dynamics of the lid have now been characterized using (19)F NMR spectroscopy with a CF(3)-labeled enzyme and CF(3)O-labeled ligands. From two-dimensional (19)F-(19)F NMR exchange spectroscopy, interconversion rates between the free and bound states of a CF(3)O-substituted tyrosine (45 ± 10 s(-1)) and the protein label (40 ± 3 s(-1)) are very similar and suggest a correlation between ligand binding and conformational reorganization of the lid. Both occur at rates that are ∼100-fold faster than turnover, and therefore these steps do not limit catalysis. A simple CF(3)O-labeled phenol also binds to the active site and induces a conformational change in the lid segment that was not previously detectable by crystallography. Exchange rates of the ligand (130 ± 20 s(-1)) and protein (98 ± 8 s(-1)) in this example are faster than those above but remain self-consistent to affirm a correlation between ordering of the lid and binding of the ligand. Both ligands also protect the protein from limited proteolysis, as expected from their ability to stabilize a compact lid structure. However, the minimal turnover of simple phenol substrates indicates that such stabilization may be necessary but is not sufficient for efficient catalysis.