Identification of the Catalytic Base of Human Aorta Lysyl Oxidase
Authors:Caitlin Ghilarducci, Jeanette Limones
Mentor:Karlo Lopez, Assistant Professor of Biochemistry, California State University, Bakersfield
Lysyl oxidase (LOX) is a copper-dependent amine oxidase responsible for the final crosslinking step of collagen in connective tissues and is also involved in many other biological processes. The active-site of LOX has been determined to have a strong central core requiring high concentrations of urea to solubilize. The enzyme is dependent on a self-processed catalytic cofactor (lysyl tyrosyl quinone, LTQ) and one copper ion. Histidine 303 (human) has been postulated to be the catalytic base necessary for LOX activity. The goals of this project were to make LOX soluble in aqueous buffers without the need of urea and also to identify the catalytic base required for LOX activity. These goals were addressed by making two key changes in LOX. First, the Nus-A solubility tag was added to the N-terminus of the enzyme and, second, Histidine 303 was mutated to isoleucine. The data indicates that with the H303I mutation, Nus-A tagged lysyl oxidase has no detectable catalytic activity as compared to the Nus-A tagged wildtype (0.11 U/mg). This is consistent with the removal of the catalytic base. In order to verify that the lack of activity was due to the removal of the catalytic base and not a misfold in the protein, LTQ was detected by western transfer using redox cycling, and total copper incorporation was quantified to 68% which is consistent with published data. Given these results, it is postulated that Histidine 303 is in fact the catalytic base necessary for LOX activity.