Evaluation by Site-Directed Mutagenesis of Aspartic Acid Residues in the Metal Site of Pig Heart NADP-Dependent Isocitrate Dehydrogenase

Abstract

Pig heart NADP-dependent isocitrate dehydrogenase requires a divalent metal cation for catalysis. On the basis of affinity cleavage studies [Soundar and Colman (1993) J. Biol. Chem. 268, 5267] and analysis of the crystal structure of E. coli NADP-isocitrate dehydrogenase [Hurley et al. (1991) Biochemistry 30, 8671], the residues Asp(253), Asp(273), Asp(275), and Asp(279) were selected as potential ligands of the divalent metal cation in the pig heart enzyme. Using a megaprimer PCR method, the Asp at each of these positions was mutated to Asn. The wild-type and mutant enzymes were expressed in Escherichia coli and purified. D253N has a specific activity, K(m) values for Mn(2+), isocitrate, and NADP, and also a pH-V(max) profile similar to those of the wild-type enzyme. Thus, Asp(253) is not involved in enzyme function. D273N has an increased K(m) for Mn(2+) and isocitrate with a specific activity 5% that of wild type. The D273N mutation also prevents the oxidative metal cleavage seen with Fe(2+) alone in the wild-type enzyme. As compared to wild type, D275N has greatly increased K(m) values for Mn(2+) and isocitrate, with a specific activity <0.1% that of wild type, and a large increase in pK(a) for the enzyme-substrate complex. D279N has only small increases in K(m) for Mn(2+) and isocitrate, but a specific activity <0.1% that of wild type and a major change in the shape of its pH-V(max) profile. These results suggest that Asp(273) and Asp(275) contribute to metal binding, whereas Asp(279), as well as Asp(275), is critical for catalysis. Asp(279) may function as the catalytic base. Using the Modeler program of Insight II, a structure for porcine NADP-isocitrate dehydrogenase was built based on the X-ray coordinates of the E. coli enzyme, allowing visualization of the metal-isocitrate site.

Identification of Mn2+-binding Aspartates from α, β, and γ Subunits of Human NAD-dependent Isocitrate Dehydrogenase

“…However, the K m values for Mn 2ϩ , isocitrate, and NAD are normal. This pattern is similar to the properties of mutants of Asp-279 of the porcine NADP-IDH (19,29), which is a "second shell" ligand of the metal ion. If ␣-Asp-234 has a corresponding location in the human NAD-IDH, replacement of this aspartate can indirectly affect the orientation of the substrate at the active site by changing the interaction of amino acid 234 with water coordinated to the Mn 2ϩ .…”

“…Thus, perturbation of the manganese-isocitrate binding by changing a ligand of the metal ion may indirectly have an adverse effect on the affinity of the enzyme for NAD. These characteristics are similar to those of mutants of Asp-275 of the porcine NADP-IDH (19,29).…”

“…Asp-279 of the B subunit is located a little further from the Mn 2ϩ but is within hydrogen bonding distance of the two water molecules that are coordinated to the metal ion (10,19). The roles of Asp-252, Asp-275, and Asp-279 of the porcine NADP-specific isocitrate dehydrogenase have been demonstrated by site-directed mutagenesis (19,29).…”

Molecular mechanisms of “off-on switch” of activities of human IDH1 by tumor-associated mutation R132H

“…As shown in Table 2, the V max and K m (ICT) values of the wild-type IDH1 are comparable to that of PmIDH and E. coli NADP-IDH (EcIDH) [22][23][24][25][26]. Compared with the wild-type IDH1, the homodimeric R132H mutant manifests a 200-fold increase in the K m whereas its specific activity is decreased to about 5%; similarly, the K m of the R132A mutant is also substantially increased by 106-fold while its specific activity is decreased to about 18% ( Table 2).…”

“…There is discrepancy in the IDH activity of the R132H mutant as Zhao et al [12] showed a 2-fold decrease in the catalytic rate while Dang et al [13] reported a 1000-fold reduction, and our data are in better agreement with that reported by Zhao et al In addition, the kinetic parameters of the R132C, R132S, and R132L mutants are similar to that of the R132H mutant ( Table 2). The Y139A and K212A mutants display < 1% activity ( Table 2), indicating that Tyr139 and Lys212, like their counterparts in EcIDH and PmIDH [22,25,27], play critical roles in the catalytic reaction. Our data also show that mutations D252A and D275A have severe effects on the K m and/or k cat due to their involvements in both ICT binding and catalysis (Table 2).…”