Catalase-peroxidases (KatG) are bifunctional hemoproteins possessing both catalase and peroxidase activities. The crystal structures of different KatGs each revealed the presence of a novel active site modification comprised of two covalent bonds between three amino acid side chains: Trp107, Tyr229, and Met255 (Mycobacterium tuberculosis numbering). Absent from the peroxidases, this Met-Tyr-Trp ‘crosslink' has been suggested to impart catalase activity to the KatGs, although how it carries out this function has been the subject of recent debate.

To better understand the role which the crosslink plays in enzyme catalysis, we have expressed several KatG(Tyr229UAA) mutants, where UAA represents the following unnatural amino acids: p-Ac, p-NH₂, p-N₃, p-Br, p-I, and p-OMe derivatives of phenylalanine. Notably, these mutants exhibited up to a 300-fold increase in peroxidase catalytic efficiency (k_cat/K_m) compared to the WT enzyme (see Table). Specifically, k_cat was enhanced by ~20-60 fold, whereas K_m was improved slightly (~1-8 fold), indicating that the gain in catalytic efficiency is mainly from increasing the rate of the peroxidase reaction, with only a minor contribution from improving substrate binding. While these KatG mutants were unable to form Met-UAA-Trp crosslinks, resulting in loss of catalase activity, they nevertheless represent one of the first examples in hemoprotein engineering where an enzymatic activity has been enhanced through the use of UAAs. Details of these and our complementary stopped-flow UV-visible spectroscopic studies will be presented.