Characterization of the Binding of Isoniazid and Analogues to Mycobacterium tuberculosis Catalase-Peroxidase
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- 作者：Xiangbo Zhao ; Shengwei Yu ; Richard S. Magliozzo
- 刊名：Biochemistry
- 出版年：2007
- 出版时间：March 20, 2007
- 年：2007
- 卷：46
- 期：11
- 页码：3161 - 3170
- 全文大小：204K
- 年卷期：v.46,no.11(March 20, 2007)
- ISSN：1520-4995
文摘
The first-line antituberculosis drug isonicotinic hydrazide (INH) is a prodrug whose bactericidalfunction requires activation by Mycobacterium tuberculosis catalase-peroxidase (KatG) to produce anacyl-NAD adduct. Peroxidation of INH is considered a required catalytic process for drug action. Thebinding of INH and a series of hydrazide analogues to resting KatG was examined using optical andcalorimetric techniques to provide thermodynamic parameters, binding stoichiometries, and kinetic constants(on and off rates). This work revealed high-affinity binding of these substrates to a small fraction offerric enzyme in a six-coordinate heme iron form, a species most likely containing a weakly bound watermolecule, which accumulates during storage of the enzyme. The binding of hydrazides is associated witha large enthalpy loss (>100 kcal/mol); dissociation constants are in the range of 0.05-1.6 
s/entities/mgr.gif">M, and opticalstopped-flow measurements demonstrated kon values in the range of 0.5-27 × 103 M-1 s-1 with verysmall koff rates. Binding parameters did not depend on pH in the range 5-8. High-affinity binding of INHis disrupted in two mutant enzymes bearing replacements of key distal side residues, KatG[W107F] andKatG[Y229F]. The rates of reduction of KatG Compound I by hydrazides parallel the on rates for associationwith the resting enzyme. In a KatG-mediated biomimetic activation assay, only isoniazid generated ingood yield the acyl-NAD adduct which is considered a key molecule in INH action, providing a betterunderstanding of the action mechanism of INH.