Evaluating the Novel Role of Tryptophan 438 in Active Turnover of Mycobacterium tuberculosis Catalase-Peroxidase

Catalase-peroxidase (KatG) is an enzyme capable of utilizing both catalase and peroxidase activity to break down hydrogen peroxide. Catalase activity by the enzyme generally dominates, but peroxidatic electron donors have been shown to stimulate catalase activity, likely by rescuing catalase-inactive intermediates resulting from off-pathway electron transfer. To investigate tryptophan 438 as a potential conduit for this misdirected transfer, a variant form of the enzyme (W438F), which contained a phenylalanine in place of the tryptophan, was produced by site-directed mutagenesis, expressed, and purified. Catalase a n d p e r o x i d a s e a c t i v i t i e s were measured v ia UV-Vis spectrophotometry, and catalase activity was also monitored by oxygen production. We observed a threefold increase in catalase activity by the variant as compared to wild-type KatG. Additionally, W438F displayed a threefold decrease in peroxidase activity. These results are consistent with the possibility that off-pathway electron transfer could occur by this route, as the phenylalanine substitution would obstruct this oxidizable passage and cause a diminished requirement for the peroxidatic rescue event. However, oxygen production data also revealed stimulation of catalase activity by peroxidatic electron donors at pH 5, and further investigation is needed to understand this pathway.