Thesis advisor: Evan R. Kantrowitz / Nucleotides, the building blocks for nucleic acids, are essential for cell growth and replication. In E. coli the enzyme responsible for the regulation of pyrimidine nucleotide biosynthesis is aspartate transcarbamoylase (ATCase), which catalyzes the committed step in this pathway. ATCase is allosterically inhibited by CTP and UTP in the presence of CTP, the end products of the pyrimidine pathway. ATP, the end product of the purine biosynthetic pathway, acts as an allosteric activator. ATCase undergoes the allosteric transition from the low-activity and low-affinity T state to the high-activity and high-affinity R state upon the binding of the substrates. In this work we were able to trap an intermediate ATCase along the path of the allosteric transition between the T and R states. Both the X-ray crystallography and small-angle X-ray scattering in solution clearly demonstrated that the mutant ATCase (K164E/E239K) exists in an intermediate quaternary structure shifted about one-third toward the canonical R structure from the T structure. The structure of this intermediate ATCase is helping to understand the mechanism of the allosteric transition on a molecular basis. In this work we also discovered that a metal ion, such as Mg2+, was required for the synergistic inhibition by UTP in the presence of CTP. Therefore, the metal ion also had significant influence on how other nucleotides effect the enzyme. A more physiological relevant model was proposed involving the metal ion. To better understand the allosteric transition of ATCase, time-resolved small-angle X-ray scattering was utilized to track the conformational changes of the quaternary structure of the enzyme upon reaction with the natural substrates, PALA and nucleotide effectors. The transition rate was increased with an increasing concentration of the natural substrates but became over one order of magnitude slower with addition of PALA. Addition of ATP to the substrates increased the rate of the transition whereas CTP or the combination of CTP and UTP exhibited the opposite effect. In this work we also studied E. coli dihydroorotase (DHOase), which catalyzes the following step of ATCase in the pyrimidine biosynthetic pathway. A virtual high throughput screening system was employed to screen for inhibitors of DHOase, which may become potential anti-proliferation and anti-malarial drug candidates. Upon the discovery of the different conformations of the 100's loop of DHOase when substrate or product bound at the active site, we've genetically incorporated an unnatural fluorescent amino acid to a site on this loop in the hope of obtaining a better understanding of the catalysis that may involve the movement of the 100's loop. / Thesis (PhD) — Boston College, 2012. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Chemistry.

Thesis advisor: Evan R. Kantrowitz / The enzyme aspartate transcarbamoylase (ATCase) is an enzyme in the pyrimidine nucleotide biosynthetic pathway. It was once an attractive target for anti-proliferation drugs but has since become a teaching model due to kinetic properties such as cooperativity and allostery exhibited by the Escherichia coli form of the enzyme. ATCase from E. coli has been extensively studied over that last 60 years and is the textbook example of allosteric enzymes. Through this past research it is understood that ATCase is allosterically inhibited by CTP, the end product of pyrimidine biosynthesis, and allosterically activated by ATP, the end product of the parallel purine biosynthetic pathway. Part of the work discussed in this dissertation involves further understanding the catalytic properties of ATCase by examining an unregulated trimeric form from Bacillus subtilis, a bacterial ATCase that more closely resembles the mammalian form than E. coli ATCase. Through X-ray crystallography and molecular modeling, the complete catalytic cycle of B. subtilis ATCase was visualized, which provided new insights into the manifestation of properties such as cooperativity and allostery in forms of ATCase that are regulated. Most of the work described in the following chapters involves understanding allostery in E. coli ATCase. The work here progressively builds a new model of allostery through new X-ray structures of ATCase*NTP complexes. Throughout these studies it has been determined that the allosteric site is bigger than previously thought and that metal ions play a significant role in the kinetic response of the enzyme to nucleotide effectors. This work proves that what is known about ATCase regulation is inaccurate and that currently accepted, and taught, models of allostery are wrong. This new model of allostery for E. coli ATCase unifies all old and current data for ATCase regulation, and has clarified many previously unexplainable results. / Thesis (PhD) — Boston College, 2013. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Chemistry.