Substrates regulate the phosphorylation status of nitrate reductase

Abstract

The effect of substrates on the phosphorylation status of nitrate reductase (NR; EC 1.6.6.1) was studied. The enzyme was obtained from the first leaf of 7‐day‐old oat (Avena sativa L. cv. Suregrain) plants, grown in the light. When desalted crude extracts were incubated with ATP, NR was strongly phosphorylated, as evidenced by the inhibition of the enzyme's activity in the presence of Mg2+. NR sensitivity to Mg2+ remained unchanged when 10 mM nitrate was added to crude extracts after ATP. Addition of nitrate before or simultaneously with ATP slightly decreased Mg2+ inhibition of NR, which was strongly diminished in the presence of 10 mM NO3−+ 100 µM NADH. Incubation with NADH alone did not affect the enzyme's susceptibility to Mg2+ inhibition. When ammonium sulfate was added to crude extracts, NR was recovered in a 0‐40% saturation fraction (F1).After incubation of F1 with ATP, the sensitivity of the enzyme to Mg2+ inhibition remained low, but it strongly increased after mixing F1 with a 45‐60% saturation fraction (F2) suggesting that also in oats an additional factor (inactivating protein, IP), which probably binds to phospho‐NR, would be required to keep the phosphorylated enzyme inactive in a +Mg2+ medium. Addition of 10 mM NO3−+ 100 µM NADH together with desalted F2 did not prevent Mg2+ inhibition suggesting that NO3− did not interfere with IP binding to phospho‐NR. Again, incubation of F1 with both substrates during in vitro phosphorylation kept the enzyme active after adding F2, even in the presence of Mg2+, After in vitro phosphorylation, NR in crude extract was hardly reactivated when incubated alone or in the presence of 10 mM NO3− at 30°C. On the other hand, a strong and very rapid reactivation was found when the extract was incubated with both nitrate and NADH. Microcystine, an inhibitor of types 1 and 2A phosphoprotein phosphatases, inhibited the reactivation of phospho‐NR induced by the substrates.The results presented here show that the substrates could prevent NR phosphorylation and induce the enzyme's dephosphorylation, but they were effective only after their binding to the NR protein. Thereby, they seemed to affect the NR protein itself and not the phosphatase‐ or the kinase‐proteins. It has been reported that nitrate binding to the enzyme's active site induces conformational changes in the NR protein. We propose that this conformational change would prevent NR phosphorylation, by converting the enzyme into a form in which the site recognized by the protein kinase is no longer accessible, and, simultaneously, stimulate NR dephophorylation by allowing the specific phosphatases to recognize NR.