| Abstract
| - Isoniazid is an antituberculosis prodrug that requires activation by the catalase-peroxidase (KatG)of Mycobacterium tuberculosis. The activated species, presumed to be an isonicotinoyl radical, couples toNADH forming an isoniazid−NADH adduct that ultimately confers antitubercular activity. We have comparedthe catalytic properties of three KatGs associated with isoniazid resistance (resistance mutation KatGs,RMKatGs: R104L, H108Q, S315T) to wild-type enzyme and two additional lab mutations (wild-type phenotypeKatGs, WTPKatGs: WT KatG, Y229F, R418L). Neither catalase nor peroxidase activities, nor the presence/absence of the Met-Tyr-Trp cross-link (as probed by LC/MS on tryptic digests of the protein), exhibited anycorrelation with isoniazid resistance. The yields of isoniazid−NADH adduct formed were determined to be1−5, 4−12, and 20−70-fold greater for the WTPKatGs than the RMKatGs for the compound I, II, and IIIpathways, respectively, strongly suggesting a role for oxyferrous KatG (supported by superoxide consumptionmeasurements) that correlates with drug resistance. Stopped-flow UV−visible spectroscopic studies revealedthat all KatGs were capable of forming both compound II and III intermediates. Rates of compound II decaywere accelerated 4−12-fold in the presence of isoniazid (vs absence) for the WTPKatGs but were unaffectedby the drug for the RMKatGs. A mechanism for isoniazid resistance which accounts for the observed reactivityfor each of the compound I, II, and III intermediates is proposed and suggests that the compound III pathwaymay be the primary factor in determining overall isoniazid resistance by specific KatG mutants, withsecondary contributions arising from the compound I and II pathways.
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