McKenna Albers Butler University
Faculty Sponsor(s): Geoffrey Hoops Butler University, Mark Macbeth Butler UniversityTM0077 is an acetyl esterase from the CE7 family and is produced by the organism Thermatoga maritima. With a highly conserved active site, the residue Proline 228 (P228) strongly contributes to the unique structure and rigidity of the active site, resulting in a high substrate specificity towards small acyl groups of TM0077. In this project, mutants of TM0077 at the P228 site were overexpressed in E. coli cells, purified, and used to analyze enzyme kinetics and substrate specificity. Variants of TM0077 were screened against 25 fluorogenic substrates to create Michaelis-Menten curves and obtain important kinetic values such as the Km, kcat and the kcat/Km. Out of the fourteen total variants analyzed, six were found to be significantly active with altered substrate specificity. The P228A mutant was found to have the broadest substrate specificity, and was uniquely efficient in the hydrolysis of some cyclic substrates. The P228I and P228G mutants also had a broader substrate specificity than the wild type, but did not improve on catalytic efficiency in all but one case. The P228V mutant exhibited high activity against straight chain, oxygenated substrates as well as the oxazole substrate. The P228Y was the most active against the simplest acetyl substrate, and also significantly active in the hydrolysis of one particular oxygenated substrate. Finally, the P228T mutant exhibited high activity with oxygenated substrates, as well as a handful of a few other unique substrates.
Biochemistry & Molecular Biology
When & Where
Gallahue Hall 108