The interaction of shikimic acid and protein phosphorylation with PEP carboxylase from the C4 dicot Amaranthus viridis.

Shikimic acid has been described as a potent competitive inhibitor of the activity of C4 phosphoenolpyruvate carboxylase (PEPC) from Amaranthus viridis. In the present study, the effects of shikimic acid were examined further with the dephospho (dark-form) and in vitro phosphorylated forms of homogeneous PEPC from A. viridis. Kinetic analysis showed that the inhibitor effect of shikimic acid was dependent on the phosphorylation state of the enzyme. Thus, the I50 value of shikimic acid for dark-form PEPC was six times lower than that for the phosphorylated enzyme (12 vs 71 microM, respectively). When Glc6P, an activator of C4 PEPC, was present in the assay medium, the I50 value increased 2- and 3-times with the phospho and dephospho PEPC-forms, respectively. Shikimic acid also markedly decreased 32P incorporation from Mg[gamma-32P]ATP into the dark-form of C4 PEPC, but not casein, catalyzed by protein kinase A. In this way, shikimic acid mimics the behaviour of L-malate, a well-known inhibitor of PEPC, in that it decreases both the enzyme's activity and phosphorylatability. Based on these data, a possible role for shikimic acid in the regulation of PEPC activity in plants is suggested.

Flavonoids as inhibitors of NADP-malic enzyme and PEP carboxylase from C4 plants.

The inhibitory effects of flavonoids on the activity of two photosynthetic enzymes such as phosphoenolpyruvate carboxylase (PEPCase) and NADP-dependent malic enzyme (NADP-ME) were evaluated. The glycosylation of hydroxyl groups on the flavonoids resulted in compounds that behaved as gradually weaker inhibitors with increased size of the substituent. Quercetin and baicalein showed a competitive inhibition pattern vs. NADP+ for NADP-ME, and a similar model for both flavonoids vs. phosphoenolpyruvate (PEP) was observed when tested on PEPCase. K(i) for NADP-ME inhibition at pH 7.0 were 0.83 microM and 1.54 microM for quercetin and baicalein, respectively. K(i) for PEPCase inhibition were 0.17 microM and 0.79 microM (quercetin and baicalein, respectively), indicating that these compounds are the most potent inhibitors described for this carboxylase. I50 values for these and other flavonoids were in the micromolar range. A tentative physiological role for the inhibitory effects observed on PEPCase is discussed.

Interaction of analogues of substrate with NADP-malic enzyme from maize leaves.

The effect of structural analogues of L-malate was studied on NADP-malic enzyme purified from Zea mays L. leaves. Among the compounds tested, the organic acids behaved as more potent inhibitors at pH 7.0 than at pH 8.0, suggesting that the dimeric form was more susceptible to the inhibition than the tetrameric form of the enzyme.Oxalate, ketomalonate, hydroxymalonate, malonate, oxaloacetate, tartrate, α-hydroxybutyrate, α-ketobutyrate, α-ketoglutarate and α-hydroxyglutarate exhibited linear competitive inhibition with respect to the substrate L-malate at pH 8.0. On the other hand, glyoxylate and glycolate turned out to be non-competitive inhibitors, while glycolaldehyde, succinate, fumarate, maleate and ß- and γ-hydroxybutyrate had no effect on the enzyme activity, at the concentrations assayed. These results suggest that the extent of inhibition was dependent on the size of the analogues and that the presence of an 1-carboxyl group along with a 2-hydroxyl or 2-keto group was important for binding of the substrate analogue to the enzyme.