An Enzymatic Study of the Role of Tyr415 in Native Catalase HPII Using the UAA Mutants: 3-Cl-Tyr415 and 3-Br-Tyr415

Co-Investigators: Reid Kinser, Megan Iverson, and Gavin RodgersCatalase HPII from Escherichia coli, is a mono-functional catalase responsible for preventing the formation of reactive oxygen species in the cell. Its active site is representative of most mono-functional catalases, making it a good model for mechanistic studies. The role of the heme-binding tyrosine was investigated by replacing Tyr415 with two types of unnatural amino acids: 3-chlorotyrosine and 3-bromotyrosine. The designed mutants were expressed and purified in large quantities alongside the native catalase with an approximate 67% purity for 3-Cl-Tyr-415 and 78.5% for 3-Br-Tyr-415. In addition, the translational machinery was shown to be high fidelity. Quantitative kinetic assays were performed on the native catalase and 3-Cl-Tyr415 and 3-Br-Tyr415 mutants. Electronic properties were unable to be investigated due to issues with purity, but the mutants were shown to have comparable activity levels to the native. In addition, they demonstrated higher turnover numbers and lower specificity constants as compared to native. Lastly, the mutants denatured at around the same temperature as the native but showed greater constancy in terms of activity over lower temperatures. Successful incorporation of a mutant at Tyr415 has never been done before in literature and possesses serious implications for the ability of scientists to understand the catalytic role of the tyrosine ligand in monofunctional catalases