Yeast cytosine deaminase (yCD) catalyzes the conversion of the prodrug 5-fluorocytosine (5FC) to 5-fluorouracil (5FU), which is a widely used drug in the treatment of cancers. yCD is of significant biomedical interest because of its potential application in combination with 5FC in gene-directed enzyme prodrug therapy (GDEPT). In this thesis, the catalytic mechanism of yCD was elucidated by both experimental and computational methods. First, the transition state structure was accessed experimentally by kinetic isotope effects (KIEs) studies at natural abundance by 13C nuclear magnetic resonance (NMR) spectroscopy. The results indicate that the reaction proceeds by a stepwise AN+DN mechanism, with the transition state in the C4-N4 bond cleavage step. Second, the catalytic mechanism of the deamination of 5-fluorocytosine (5FC) was investigated by an ONIOM (B3LYP/6-31G*:Amber)-electronic embedding scheme and was compared side-by-side with that of cytosine. The higher catalytic efficiency (kcat/Km) for 5FC is likely due to the energetically more favorable intermediates formed during the reaction. The method alone was not suitable to elucidate the OZn-Zn bond breakage because relatively large rearrangements of the active site residues are required. Hence, Quantum Mechanics/Molecular Mechanics (QM/MM) MD simulations were set up to incorporate configurational sampling into chemical transformation. In the reaction pathway proposed, it is the C4-N4 bond cleavage step, not the OZn-Zn bond cleavage step, that is the rate-limiting step for the overall reaction. Functions of conserved residues around the active site were investigated by site-directed mutagenesis, biochemical, NMR, and computational methods. Glu64 is found to be essential in catalysis due to its role as a general base/acid. Steady-state kinetic studies show that mutating Glu64 to Ala causes a dramatic decrease in kcat by a factor of >3000. Glu64 also participates in the stabilization of the intermediates and transition state, suggested by 19F NMR experiments and ONIOM calculations. Asn51 and Asp155 interact with the substrate through hydrogen bonding, by which the substrate is anchored in the right position during the initial binding and at the transition state. These residues are important for substrate binding and catalysis.yCD belongs to the cytidine deaminase (CDA) family of purine/pyrimidine deaminases. Enzymes in this family have similar three-dimensional structures with a zinc-containing catalytic apparatus. Studies on yCD not only elucidate the enzymatic mechanism of yCD itself, but also provide significant insights into the catalytic mechanism of enzymes in this family. Moreover, these findings can facilitate antimicrobial drug design and gene directed therapy against cancers.
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