Doctoral Dissertations

Date of Award

8-2019

Degree Type

Dissertation

Degree Name

Doctor of Philosophy

Major

Life Sciences

Major Professor

Pratul Agarwal

Committee Members

Engin Serpersu, Hong Guo, Xiaolin Cheng

Abstract

The enzymes of the ribonuclease (RNase) family catalyze the hydrolysis of ribonucleic acid (RNA). There is a wide interest in therapeutic interventions of human RNases (hRNases) due to their critical role in host defense, cancer cell proliferation and neurodegenerative diseases. Development of structure-based therapeutics targeting individual members of a closely related enzyme family is difficult. The structural conservation among hRNases cannot explain a million-fold difference in the catalytic efficiency of these enzymes. We hypothesize, this ambiguity in the structure activity relationship of hRNases can be explained by their dynamical behavior. The rate of substrate turnover in RNases correlates strongly with the rates of conformational dynamics. Moreover, catalytic efficiency is also linked to the ability of an enzyme to sample conformational sub-states that promote specific interaction between reactants at various stages of catalysis. Detailed characterization of the structure-function-dynamics relationship in hRNases can help in determining similarities and differences in the motions associated to catalysis thereby enhancing the possible druggability of this enzyme family. This study investigates the role of functionally relevant conformational sub-states and dynamics in each step of the RNase members catalytic cycle; apo, substrate-bound, the chemical step, and product release. Computer simulations and nuclear magnetic resonance (NMR) relaxation dispersion experiments indicated an increased dynamics in distal loop regions of RNases in their apo state. Similar dynamical patterns were observed within the members of an individual sub-family, while dynamics were different across sub-families. Nucleotide binding properties probed using computer simulations indicated diverse binding preferences in hRNases. Additionally, structural and dynamical properties of hRNases varied significantly with subtle change in temperature. Quasi anharmonic analysis revealed sampling of separate conformational sub-states in product release step of RNases. Further, NMR chemical shift titrations along with the chemical shift projection analysis revealed distinct conformational rearrangements in hRNases upon binding of ligands that mimic cleaved products. Steady-state kinetics experiments showed million-fold difference in the catalytic efficiency of hRNases. Finally, hybrid quantum mechanical/molecular mechanics calculations identified conformational sub-states associated with the chemical step in bovine RNase A. Broadly, these results strongly support our hypothesis that dynamics modulates catalytic efficiencies of structurally related enzyme super-families like RNases.

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