Abstract
The steady-state kinetics of the NADPH + FAD-dependent reduction of nitrate by nitrate reductase from Penicillium chrysogenum was studied at pH 6.18. At this sub-optimum pH, V max was about 83 units × mg protein −1 compared with 225 units × mg protein −1 at pH 7.20. All initial velocity reciprocal plot patterns at pH 6.18 as well as the NADP +/nitrate product inhibition pattern were intersecting. In contrast, the NADP(H)/nitrate plots at pH 7.20 were parallel ( Renosto, F. et al. J. Biol. Chem. 256 , 8616, 1981 ). A major effect of lowering the assay pH was to change the K m for FAD from 0.17 μM at pH 7.20 to 4 μM at pH 6.18. The results suggest that nitrate reductase has a steady-state random kinetic mechanism in which k cat in the forward direction at pH 7.20 (ca. 375 sec −1) is greater that k off for the dissociation of one or more substrates. Several observations suggest that k off for FAD is extremely small at pH 7.20.
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