Kinetic mechanism of assimilatory NADH:nitrate reductase from Chlorella

Chlorella nitrate reductase catalyzes the reduction of nitrate to nitrite by NADH. Initial velocity studies showed that the kinetic mechanism is sequential, indicating that both substrates must bind to the enzyme before any products are released. Product inhibition with NAD and nitrite showed that competitive inhibition was observed when the inhibitor was similar to the varied substrate, while noncompetitive inhibition was observed when the inhibitor was dissimilar to the varied substrate. Likewise, dead-end inhibition with adenosine 5'-diphosphoribose and thiocyanate showed competitive inhibition when the inhibitor was similar to the varied substrate and noncompetitive inhibition when the inhibitor was dissimilar to the varied substrate. These results indicate that Chlorella nitrate reductase follows a random bi bi kinetic mechanism. Phosphate was found to stimulate NADH:nitrate reductase activity and 2-fold. The NADH:cytochrome c reductase activity associated with nitrate reductase was not affected by phosphate suggesting the effect of phosphate is on the nitrate-reducing moiety of the enzyme. Phosphate increases Vmax but has no effect on the apparent Km for nitrate.