The regulation and catalytic mechanism of the NADP-malic enzyme from tobacco leaves

Abstract

The non-photosynthetic NADP-malic enzyme EC 1.1.1.40 (NADP-ME), which catalyzes the oxidative decarboxylation of L-malate and NADP + to produce pyruvate and NADPH, respectively, and which could be involved in plant defense responses, was isolated from Nicotiana tabacum L. leaves. The mechanism of the enzyme reaction was studied by the initial rate method and was found to be an ordered sequential one. Regulation possibilities of purified cytosolic NADP-ME by cell metabolites were tested. Intermediates of the citric acid cycle (α-ketoglutarate, succinate, fumarate), metabolites of glycolysis (pyruvate, phosphoenolpyruvate, glucose-6-phosphate), compounds connected with lipogenesis (coenzyme A, acetyl-CoA, palmitoyl-CoA) and some amino acids (glutamate, glutamine, aspartate) did not significantly affect the NADP-ME activity from tobacco leaves. In contrast, macroergic compounds (GTP, ATP and ADP) were strong inhibitors of NADP-ME; the type of inhibition and the inhibition constants were determined in the presence of the most effective cofactors (Mn 2+ or Mg 2+ ), required by NADP-ME. Predominantly non-competitive type of inhibitions of NADP-ME with respect to NADP + and mixed type to L-malate were found.

Enzyme Regulation during Plant Stress

“…Final metabolic products or energetically rich compounds such as ATP and GTP often serve as inhibitors of enzymes. In the case of tobacco NADP-ME was found that this enzyme is inhibited by GTP, ATP, and ADP [11].…”

Tobacco Hatch-Slack Enzymes are Involved in Both, Abiotic and Biotic Stress Response

“…The regulation of the above mentioned enzymes is also interesting [13][14][15][16][17]. While the tobacco leaves NADP-ME was probably synthesized de novo during a viral infection and drought stress, the regulation of PEPC under these conditions seems to be affected by phosphorylation, which increases the enzyme activity and sensitivity to the activator D-glucose-6-phosphate [10,12,13].…”