Ascaris suum NAD-Malic Enzyme Is Activated by l-Malate and Fumarate Binding to Separate Allosteric Sites

Abstract

The kinetic mechanism of activation of the mitochondrial NAD-malic enzyme from the parasitic roundworm Ascaris suum has been studied using a steady-state kinetic approach. The following conclusions are suggested. First, malate and fumarate increase the activity of the enzyme in both reaction directions as a result of binding to separate allosteric sites, i.e., sites that exist in addition to the active site. The binding of malate and fumarate is synergistic with the K(act) decreasing by >or=10-fold at saturating concentrations of the other activator. Second, the presence of the activators decreases the K(m) for pyruvate 3-4-fold, and the K(i) (Mn) >or=20-fold in the direction of reductive carboxylation; similar effects are obtained with fumarate in the direction of oxidative decarboxylation. The greatest effect of the activators is thus expressed at low reactant concentrations, i.e., physiologic concentrations of reactant, where activation of >or=15-fold is observed. A recent crystallographic structure of the human mitochondrial NAD malic enzyme [13] shows fumarate bound to an allosteric site. Site-directed mutagenesis was used to change R105, homologous to R91 in the fumarate activator site of the human enzyme, to alanine. The R105A mutant enzyme exhibits the same maximum rate and V/K(NAD) as does the wild-type enzyme, but 7-8-fold decrease in both V/K(malate) and V/K(Mg), indicating the importance of this residue in the activator site. In addition, neither fumarate nor malate activates the enzyme in either reaction direction. Finally, a change in K143 (a residue in a positive pocket adjacent to that which contains R105), to alanine results in an increase in the K(act) for malate by about an order of magnitude such that it is now of the same magnitude as the K(m) for malate. The K143A mutant enzyme also exhibits an increase in the K(act) for fumarate (in the absence of malate) from 200 microM to about 25 mM.

Identification of Domains Involved in Tetramerization and Malate Inhibition of Maize C4-NADP-Malic Enzyme

“…Site-directed mutagenesis studies indicated that 2 amino acid residues were found to be implicated in this activation: Arg-105 (homologous to R91 of human NAD(P)-ME) and Lys-143 (Fig. 4A) (17). The effect of malate on A. suum NAD-ME is opposite to the effect of this substrate on C 4 -NADP-ME (Fig.…”

“…On the other hand, Ascaris suum NAD-ME is activated by both malate and fumarate, which bind to different binding sites that act synergically (17). Site-directed mutagenesis studies indicated that 2 amino acid residues were found to be implicated in this activation: Arg-105 (homologous to R91 of human NAD(P)-ME) and Lys-143 (Fig.…”

“…This would have been the case if the malate binding site responsible for malate inhibition was located in the interface between the dimers, as is the case of the allosteric site for fumarate in the human NADP-ME (14), and, possibly, the allosteric activating site for malate in A. suum NAD-ME (17). Alternatively, if the inhibition was related to heterogeneity of the malate active binding sites in the tetramer, as was suggested for the pigeon liver NADP-ME (18,19) (see below), it was reasonable that the tetramer (C 4 -NADP-ME) was inhibited by malate, whereas the dimer (non-C 4 -NADP-ME) was not.…”

“…Alignments were restricted to those areas identified as part of the fumarate (human NAD(P)-ME) and malate and fumarate (A. suum NAD-ME) allosteric binding sites. The amino acid residues underlined and highlighted have been identified by site-directed mutagenesis to be involved in activation by fumarate and malate (14,16,17). B, residues of the region involved in the oligomerization state of maize NADP-MEs.…”

Three Different and Tissue-specific NAD-Malic Enzymes Generated by Alternative Subunit Association in Arabidopsis thaliana

“…It should be noted that the differential expression pattern observed is consistent with AtGenExpress The region with the most significant differences is underlined. The amino acid residues homologous to the residues involved in fumarate activation of human NAD(P)-ME and A. suum NAD-ME are indicated in light gray (33,41,42). The arrow indicates the site where the sequences of NAD-ME1 and -2 were exchanged for the generation of the chimeric proteins NAD-ME1q and -2q.…”