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| author | Saif Yasin |
| title | Kinetic and Computational Analysis of phosphorothioate and phosphorodithioate substitutions in metal ion catalyzed cleavage of RNA models |
| abstract | Metal ion catalysis is critical to phosphodiester cleavage; therefore, determining their underlying catalytic mechanisms is essential
for understanding phosphodiesterase function. This study utilized kinetic and computational methods to quantify the catalytic significance of metal ion interactions with
non-bridging oxygen moieties using thio-substitutions, which should diminish inner sphere coordination. Experimentally, Ca2+ catalyzed cleavage of 5-uridine,
3'-guanosine dinucleotide model (UpG), and its phosphorothioate and phosphorodithioate analogs indicated that RP and SP enantiomers display minimal
thio-effects (kO/kS) of 2.7 and 2.5, respectively, compared to the dithioate model defect of 37. These results suggest direct interactions with the
non-bridging oxygen moieties, but that a single thiosubstitution can be compensated for. Despite catalytic defects, binding did not exhibit defects. Furthermore, the lack
of catalysis provided by Co(NH3)63+, an outer sphere model, highly supports our hypothesis of inner sphere coordination. Density Functional
Theory computations on an RNA model suggest inner sphere coordination would exhibit both binding and catalytic defects upon thio-substitutions. While monothioate
substitutions suggest minimal structure defects, dithioate substituted RNA models exhibited energetic defects, worsened hydrogen bonding stabilization, and hindered
electrostatic interactions. Therefore, we propose that initial metal ion binding relies on outer sphere coordination, while catalysis involves direct interactions with
non-bridging oxygen atoms through inner sphere coordination. Further analysis is necessary to improve the validity of our model system, to characterize the role of the
solvation shell to catalysis and to investigate the role of outer sphere coordination in binding. Overall, thio-substitutions are found to serves as a useful model,
highlighting the importance of direct coordination to the remarkable rate enhancement provided by metal ions in phosphodiester transesterification. |
| school | The College of Liberal Arts, Drew University |
| degree | B.A. (2017) |
| advisor | Adam Cassano |
| committee | Ryan Hinrichs
Minjoon Kouh |
| full text | SYasin.pdf |
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