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This bypass mechanism52 would permit for both catalase and peroxidase activities, but might be the underlying reason for decreased levels of INH-NADH adduct formation. Generally, our outcomes right here on KatG(S315G) with t-BuOOH and H2O2 (G/GOx), which show only slightly (2-fold or much less) attenuated levels of INH-NADH adduct formation, are in agreement with those previously observed by Magliozzo and coworkers for this mutant,52 who also made use of the enzymatic H2O2-generating method. Of importance, on the other hand, is the fact that when utilizing the superoxide-generating technique xanthine/xanthine oxidase within this study, no detectable quantity of the INH-NADH adduct was formed by KatG(S315G) (0 lM), in comparison with 32 lM for WT KatG. As a result, we hypothesize that a superoxide-dependent pathway for INH activation could Dentified a household of Orf recombinases from diverse lambdoid phages sharing possibly be a single achievable explanation for the INH-resistance observed for Mtb isolates that harbor this mutation. We surmise that this mutation completely disrupts the capacity of the enzyme to utilize superoxide as an oxidant involved in INH activation, though the molecular details of this disruption are unclear at thisCade et al.PROTEIN SCIENCE VOL 19:458--time. A single possibility is that oxyferrous KatG(S315G) is unable to oxidize INH, comparable to our observations with KatG(S315T),14,28 for which we recommended that the underlying reason for the observed final results could be mutation-induced (1) disruption with the active web-site hydrogen-bonding network, (2) variations in heme or side-chain redox potential, (3) increased stability from the oxyferrous species rendering it inactive, or (4) lowered affinity for INH, although this S (1 mg/ml) were applied to a 24 ml Superose 12 HR 10/30 column. latter possibility is not supported by the INH-binding studies for KatG(S315G).52 It may also be that the bypass mechanism suggested by Magliozzo and coworkers52 for Compounds I/II circumvents oxyferrous KatG(S315G) reactivity, and additional studies on the oxyferrous species within this mutant will likely be necessary to elucidate its lack of reactivity.Esults in a substantial improve in Kd (INH) of 400 lM for the mutant, in comparison to 2.5 lM for WT KatG.69 Remarkably, having said that, despite possessing an a lot more sterically-constraining Ser!Ile mutation, the mutant KatG(S315I) will not drastically attenuate (2-fold) INH-NADH adduct formation under any conditions studied. Even though these results recommend that amino acid size might not necessarily dictate substrate channel accessibility, they could also be interpreted to recommend that adduct formation might not be the only factor to consider when examining the underlying mechanisms of INH-resistance in KatG. Moreover, this mutant possesses significant catalase and peroxidase activities, and as such, loss of enzyme function cannot be correlated with INH-resistance. Additional research of the KatG(S315I) mutant, such as structural and spectroscopic investigations, will be essential to decide the components that contribute to isoniazid resistance within this mutation. Magliozzo and coworkers have lately studied the mutant KatG(S315G).52 While the Ser!Gly mutation was discovered not to disrupt INH binding (Kd 1 lM) nor alter catalase or peroxidase activities to a great extent, double-mixing stopped-flow experiments revealed that INH was a poor substrate for lowering the Compound I intermediate of KatG(S315G), and it was hypothesized that INH resistance in this mutant arose upon the shunting of catalytic intermediates typically responsible for INH activation toward other reaction pathways that didn't bring about substrate oxidation.