Date of Award

1-1-2012

Document Type

Campus Access Dissertation

Department

Chemistry and Biochemistry

Sub-Department

Chemistry

First Advisor

Lukasz Lebioda

Abstract

Part I:

N10-Formyltetrahydrofolate Synthetase (FTHFS), one of several folate enzymes, catalyzes the conversion of tetrahydrofolate (THF) to N10-formyltetrahydrofolate (fTHF) in an ATP dependent manner. In some organisms it is essential for C1 carbon fixation processes for cellular biosynthesis and has an increased concentration in bacteria that perform both acetogenesis, from the Wood-Ljungdahl pathway of autotrophic CO2 fixation, and purinolysis, through the glycine synthase/reductase pathway. The formation of fTHF occurs via a two-step process, which involves the formation of a formylphosphate (XPO) intermediate. The XPO intermediate has been proposed based on kinetic and spectroscopic experiments and confirmed through structure determination, which provided insight into the mechanism that FTHFS employs. Structures of FTHFS from the thermophilic homoacetogen, Moorella thermoacetica, with two sets of intermediates: ADP/XPO and ATPγS, as well as with two inhibitory substrate analogues, folate and ZD9331/XPO were determined to 2.5 Å, 2.84 Å, 3.0 Å, and 2.67 Å resolution respectively. Additionally, the native structure, originally determined at 2.5 Å resolution, was redetermined at 2.2 Å resolution. The enzyme is a tetramer, or a dimer of dimers, composed of four identical subunits. The bound nucleosides are observed within each of the subunits, totaling four per tetramer. Folate or the folate analogue, ZD9331, occupies the same space within the active site as the nucleosides. The presence and position of substrate, substrate analog, and intermediate within the active site supports the catalytic mechanism hypothesis that FTHFS works via a random bi uni uni bi ping-pong ter ter mechanism.

Part II:

Human thymidylate synthase (hTS) is a well-validated target in the chemotherapy of colorectal cancer and some other neoplasms. It catalyzes the transfer of a methyl group from methylenetetrahydrofolate (mTHF) to deoxyuridine monophosphate (dUMP) to form deoxythymidine monophosphate (dTMP). This enzyme has two conformations of catalytic loop 181-197, which is unique among TS enzymes. One of the conformations places crucial catalytic residues outside the active site therefore rendering it inactive, while the other is similar to those found in structures of TS enzymes from other species, which is active. In wild type hTS (wthTS), the conformers are in equilibrium and equally populated. Two mutations have been designed: 1) an arginine to lysine mutation at position 163 which results in the destabilization of the inactive conformation and is therefore locked in the active conformation and 2) a methionine to lysine mutation at position 190 which yields a strong shift in the equilibrium towards the inactive conformation thus destabilizing the active conformation. The structure of R163K revealed a strong shift of the equilibrium towards the active conformation. With this mutant enzyme, crystal-soaking studies can be done which show asymmetric binding of ligands between the subunits. Structures of R163K also show differences in the environment of the catalytic Cys195, correlating to thiol reactivity of the residue. Additionally, this active mutant allows for the diffusion of drug candidates and the monitoring of their mode of interaction with the hTS target. It also allows for the diffusion and determination of the mode of binding of compounds. The M190K variant of hTS allows for the development of allosteric ligands as potential drug candidates, which will not only help stabilize the inactive conformation of hTS, but could also lead to decreased drug resistance in chemotherapy patients. Allosteric inhibition studies lead to the investigation of hTS SUMOylation and how prevention of nuclear hTS could be accomplished. Drug development for the prevention of TS-SUMO (Small Ubiquitin-like Modifier) binding would inhibit hTS translocation into the nucleus, thereby impeding base excision repair (BER). Inhibition of BER could potentially lead to apoptosis of cancer cells. Modeling the interaction of hTS with nuclear transport proteins allows for the design of other allosteric inhibitors, which is a novel approach to hTS inhibition.

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