FIG 6.

Kinetic analysis of INH-NAD product formation using a spectrophotometric assay. (a to c) IN-NAD adduct formation was followed by monitoring the characteristic signal at 326 nm. The reactions were carried out in triplicate, in 96-well plates with a total volume of 0.2 ml. Each reaction included 0.5 μM KatG^bov (a), KatG^mari (b), or KatG^avp (c) in the presence of 8 mM NAD⁺, 0.5 units glucose oxidase (Gox), 5 mM glucose, and various concentrations of INH (0.15 to 8 mM). The reaction without NAD⁺ was carried out as a control and was subtracted from the total reaction. A set of representative results with increasing INH concentrations are shown for simplicity. (d) Michaelis-Menten plot presenting the initial reaction rates of adduct formation as a function of INH concentration, as calculated from a linear fit of the first 5 min of the experiments described in (a) to (c).