Date of Award
Francisella tularensis is the bacteria responsible for causing the disease tularemia and is listed as one of the top three-biowarfare agents. Among the proteins essential to the virulence and infectivity of F.tularensis are multiple esterases, which are enzymes that break down various ester, thioester, and amide bonds. In this project, the catalytic activity, substrate speci fi city, and structure of a putative esterase from F.tularensis was studied. Latent fluorophores based on the molecule, fluorescein, were unmasked by the enzymatic activity of the esterase and the increase in fluorescence was measured over time to determine how well the e tcrase recognized different substrates. The esterase FlT258 from F. IIJlarensis activated a variety of simple latent fluorophore substrates with catalytic efficiencies ranging from 5075 M"s" for a simple propyl ester to 294.8 M' 's" for a teniary e ter. These simple substrates were recognized by the esterase with KM va lues ranging from 0.54 to 2 1.4 f,M , and sterically occluded substrates had significantly reduced kinetic turnover (kc",) compared to the simplest substrates. In addition to the wild type esterase, the kinetic a tivity of five different variants of the esterase with single amino acid mutations were characterized against two latent fluorophore substrates to determine more information about the binding pocket of the esterase. The kinetic activity of each of the variants decreased significantly from the wild-type enzyme activity and indicated that the binding pocket is fairly invariant to substitution. Activity, 3D structure, and primary structure comparisons suggest that this esterase belongs to the carboxylesterasc family. Although lillie is known about the specific biological role of FTI 0258C and other carboxylesterases from its fanlily, the promiscuity of its enzymatic eclJ\ll) ould Ix uoollO dc\ I P f'OlmlJ:tl Jru m,>dcl thaI ulIlll 1M Unl
Weston, Leigh Anna, "Characterization of esterase activity from the bacteria, Francisella tularensis, the causative agent of tularemia" (2011). Undergraduate Honors Thesis Collection. 112.