Metabolic activators of spinach leaf nitrate reductase: Effects on enzymatic activity and dephosphorylation by endogenous phosphatases

Abstract

Nitrate reductase (NR; EC 1.6.6.1) in spinach (Spinacia oleracea L.) leaves was inactivated in the dark and reactivated by light in vivo. When extracted from dark leaves, NR activity was lower and more strongly inhibited by Mg2+ relative to the enzyme extracted from leaves harvested in the light. When dark extracts were desalted at pH 6.5 and preincubated at 25° C prior to assay, enzyme activity (assayed either in the presence or absence of Mg2+) remained essentially constant, i.e. there was no spontaneous reactivation in vitro. However, addition of certain metabolites resulted in a time- and concentration-dependent activation of NR in vitro. Effective activators included inorganic phosphate (Pi), 5′-AMP, and certain of its derivatives such as FAD and pyridine nucleotides (both oxidized and reduced forms). All of the activators increased NR activity as assayed in the absence of Mg2+, whereas some activators (e.g. Pi, 5′-AMP and FAD) also reduced Mg2+ inhibition. The reduction of Mg2+ inhibition was also time-dependent and was almost completely prevented by a combination of okadaic acid plus KF, suggesting the involvement of dephosphorylation catalyzed by endogenous phosphatase(s). In contrast, the activation of NR (assayed minus Mg2+) was relatively insensitive to phosphatase inhibitors, indicating a different mechanism was involved. Compounds that were not effective activators of NR included sulfate, ribose-5-phosphate, adenosine 5′-monosulfate, coenzyme A, ADP and ATP. We postulate that NR can exist in at least two states that differ in enzymatic activity. The activators appear to interact with the NR molecule at a site distinct from the NADH active site, and induce a slow conformational change (hysteresis) that increases NR activity (assayed in the absence of Mg2+). Possibly as a result of the conformational change caused by certain activators, the regulatory phospho-seryl groups are more readily dephosphorylated by endogenous phosphatases, thereby reducing sensitivity to Mg2+ inhibition. Preliminary results suggest that light/dark transitions in vivo may alter the distribution of NR molecules between the low- and high-activity forms.