Catalytic Mechanism of Human Ten-Eleven Translocation‑2 (TET2) Enzyme: Effects of Conformational Changes, Electric Field, and Mutations

Ten-eleven translocation (TET) family of enzymes are non-heme Fe­(II)- and 2-oxoglutarate (2OG)-dependent oxygenases that perform oxidation of the methyl group of the 5-methylcytosine (5mC) on DNA. TET enzymes play a crucial role in epigenetic modifications and have been linked to malignant transformation and various forms of cancer such as prostate, lung, and breast cancer. In this study, molecular dynamic (MD) and combined quantum mechanic/molecular mechanic (QM/MM) approaches were used to explore the catalytic mechanism, conformational dynamics, and the effects of mutations during the first oxidation from 5mC substrate to 5hmC by human TET2 enzyme. The studies reveal that a correlated motion between the main structural elements in TET2, the glycine–serine (GS) linker and the Cys-rich N-terminal (Cys-N) subdomain, plays a key role in the orientation of the DNA substrate in the wild-type (WT) TET2. This correlated motion is affected in the mutant forms of TET2. The conformational changes in the WT TET2 influence the rate of the hydrogen atom abstraction (HAT) step; however, its mechanism via σ-channel remains unchanged. The results enabled us to identify key residues that are crucial for HAT and to delineate their crucial energy contributions and long-range correlated interactions. Notably, several remote mutations, far away from the TET2 enzymes’ active site, unexpectedly exercise a substantial effect on the HAT step by (i) increasing the required activation barrier and (ii) switching the electron transfer mechanism from σ- to π-channel. Remarkably, mutations alter the internal electric fields along the FeO bond that in synergy with changes in the geometric factors (e.g., the hydrogen abstraction distance and the angle) influence the reactivity of the TET2 mutant forms. The kinetic isotope effect (KIE) calculations indicate weak tunneling contributions in the WT, with variations in the mutant forms. The double-mutant form K1299E-S1303N, which has clinical implications in patients with refractory anemia, exercises a substantial effect on the activation barrier, electric field, and the KIE. This study offers a novel insight into molecular biophysics and pathology of the human TET2 enzyme and asserts the vital effects of the protein residues in the second sphere and beyond on the catalytic process.