Nitrate reductase from Penicilliumchrysogenum: Kinetic mechanism at sub-optimum pH

The steady-state kinetics of the NADPH + FAD-dependent reduction of nitrate by nitrate reductase from $\text{Penicillium}\text{chrysogenum}$ 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. $\text{et}\text{al.}$ J. Biol. Chem. $\text{256}$, 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.