Thesis advisor: Evan R. Kantrowitz / E.coli Aspartate transcarbamoylase (ATCase) is the allosteric enzyme that catalyzes the committed step of the de novo pyrimidine biosynthesis pathway. ATCase facilitates the reaction between L-aspartate and carbamoyl phosphate to form N-carbamoyl-L-aspartate and inorganic phosphate. The holoenzyme is a dodecamer, consisting of two trimers of catalytic chains, and three dimers of regulatory chains. ATCase is regulated homotropically by its substrates, and heterotropically by the nucleotides ATP, CTP, and UTP. These nucleotides bind to the regulatory chains, and alter the activity of the enzyme at the catalytic site. ATP activates the rate of ATCase's reaction, while CTP inhibits it. Additionally, UTP and CTP act together to inhibit the enzyme synergistically, each nucleotide enhancing the inhibitory effects of the other. Two classes of CTP binding sites have been observed, one class with a high affinity for CTP, and one with a low affinity. It has been theorized that the asymmetry of the binding sites is intrinsic to each of the three regulatory dimers. It has been hypothesized that the second observed class of CTP binding sites, are actually sites intended for UTP. To test this hypothesis, and to gain more information about heterotropic regulation of ATCase and signal transmission in allosteric enzymes, the construction of a hybrid regulatory dimer was proposed. In the successfully constructed hybrid, each of the three regulatory dimers in ATCase would contain one regulatory chain with compromised nucleotide binding. This project reports several attempts at constructing the proposed hybrid, but ultimately the hybrid enzyme was not attained. This project also reports preliminary work on the characterization of the catalytic chain mutant D141A. This residue is conserved in ATCase over a wide array of species, and thus was mutated in order to ascertain its significance. / Thesis (BS) — Boston College, 2006. / Submitted to: Boston College. College of Arts and Sciences. / Discipline: Chemistry. / Discipline: College Honors Program.

Phosphofructokinase from Escherichia coli (EcPFK) is allosterically regulated by MgADP and phospho(enol)pyruvate (PEP). Both molecules compete for binding to the same allosteric site, however, MgADP activates and PEP inhibits the binding of fructose-6-phosphate (F6P) to the active site. The mode by which this enzyme can differentiate between the two ligands and cause the appropriate response is important for the understanding of the basis of allosteric regulation. We studied the interactions between an active site and an allosteric site (heterotropic interactions) within the protein, and found that each of the four unique heterotropic interactions is unique and the magnitudes of the coupling free energies for MgADP activation sum up to 100% that of wildtype EcPFK without homotropic cooperativity in F6P binding. We took on the kinetic and structural characterization of phosphofructokinase from Lactobacillus bulgaricus (LbPFK) to reveal an enzyme that exhibits allosteric properties in spite of previous kinetic studies performed by Le Bras et al. (1991). We have identified residues in EcPFK (Asp59, Gly184 and Asp273), which are important for the allosteric responses to both MgADP and PEP. Interestingly, Lys214 is only important in PEP inhibition and not MgADP activation. We can also differentially disrupt the MgADP heterotropic interactions with the introduction of G184C within the protein. These results suggest that there are different pathways for allosteric communication within the enzyme: different paths for MgADP activation and PEP inhibition, and different paths for each heterotropic interaction with Gly184 being important for the 33Å MgADP heterotropic interaction.

Displaced femoral neck fractures comprise more than a third of all hip fractures. There is controversy as to the optimal treatment. Despite attempts to improve the methods for internal fixation, complication rates have been almost unchanged: 20-40% non-union and late segmental collapse in another 10-20%. Internal fixation has been the preferred treatment in Scandinavia, whereas primary hemi- or total arthroplasty have been more prevalent in the rest of Europe and North America. In this study, patients 75 years or older, including those with mental impairment, were randomized to either internal fixation or cemented primary total hip arthroplasty (THA). A total of 146 hips in 143 patients were followed for two years. After one year 23% had died, and after two years 29%. Mortality was about the same in both groups. The accumulated mortality was pronounced among the mentally impaired patients. In the internal fixation group, 44% underwent further surgery. In the THA group, 18% dislocated. The dislocation rate was higher for the mentally impaired patients. The Harris hip scores were higher in the THA group, whereas pain was more common in the internal fixation group. The first 50 patients in each treatment group were studied concerning heterotopic ossification (HO), a well-known complication after THA. The incidence of HO in the THA group was similar to what is found after THA due to osteoarthritis. However, only 1/39 developed severe symptoms. A subgroup of 100 patients was included in a study concerning nutritional status and functional capacity using the Modified Norton scale, Katz index of ADL and a questionnaire measuring instrumental activities of daily living. The THA group fared better concerning weight change over time, locomotion and pain. The nutritional intervention did not show any measurable effects. All patients were followed until two years postoperatively and all fracturerelated hospital costs, including reoperations, were calculated. We found no difference in total costs between the treatment groups. Costs to the municipality were calculated comparing the baseline cost before surgery with the average cost per month during the first postoperative year. No difference was found between the treatment groups. On the basis of our results, we recommend arthroplasty for patients in this age group with normal mental function and high functional demands.

Diss. (sammanfattning) Linköping : Univ., 2002. / Härtill 4 uppsatser.