Ser95, Asn97, and Thr78 are important for the catalytic function of porcine NADP-dependent isocitrate dehydrogenase

Abstract

The mammalian mitochondrial NADP-dependent isocitrate dehydrogenase is a citric acid cycle enzyme and an important contributor to cellular defense against oxidative stress. The Mn(2+)-isocitrate complex of the porcine enzyme was recently crystallized; its structure indicates that Ser(95), Asn(97), and Thr(78) are within hydrogen-bonding distance of the gamma-carboxylate of enzyme-bound isocitrate. We used site-directed mutagenesis to replace each of these residues by Ala and Asp. The wild-type and mutant enzymes were expressed in Escherichia coli and purified to homogeneity. All the enzymes retain their native dimeric structures and secondary structures as monitored by native gel electrophoresis and circular dichroism, respectively. V(max) of the three alanine mutants is decreased to 24%-38% that of wild-type enzyme, with further decreases in the aspartate mutants. For T78A and S95A mutants, the major changes are the 10- to 100-fold increase in the K(m) values for isocitrate and Mn(2+). The results suggest that Thr(78) and Ser(95) function to strengthen the enzyme's affinity for Mn(2+)-isocitrate by hydrogen bonding to the gamma-carboxylate of isocitrate. For the Asn(97) mutants, the K(m) values are much less affected. The major change in the N97A mutant is the increase in pK(a) of the ionizable metal-liganded hydroxyl of enzyme-bound isocitrate from 5.23 in wild type to 6.23 in the mutant enzyme. The hydrogen bond between Asn(97) and the gamma-carboxylate of isocitrate may position the substrate to promote a favorable lowering of the pK of the enzyme-isocitrate complex. Thus, Thr(78), Ser(95), and Asn(97) perform important but distinguishable roles in catalysis by porcine NADP-specific isocitrate dehydrogenase.

Figure 1.

View of Mn²⁺-isocitrate site of one subunit of porcine mitochondrial NADP-dependent isocitrate dehydrogenase based on the crystal structure (PDB 1LWD). The backbone of the B subunit is shown in cyan, while that of the A subunit is pink. The side chains of B subunit Asn⁹⁷, Ser⁹⁵, and Thr⁷⁸, as well as isocitrate bound to the B subunit are colored by atom type with green = C, white = H, blue = N, and red = O. The Mn²⁺ is green. The γ-carboxylate of isocitrate is shown hydrogen-bonded to the amide of Asn⁹⁷, as well as to the –OH of Ser⁹⁵ and Thr⁷⁸. The distances between the indicated atoms are given in Å.

Figure 2.

SDS-polyacrylamide gel electrophoresis of purified wild-type and mutant enzymes. (Lane 2) Wild-type, (lane 3) T78A, (lane 4) S95A, (lane 5) N97A, (lane 6) T78D, (lane 7) S95D, and (lane 8) N97D. Lanes 1 and 9 contain standard proteins: phosphorylase b (97,000), albumin (66,000), ovalbumin (45,000), carbonic anhydrase (30,000), trypsin inhibitor (20,100), and α-lactalbumin (14,400). Approximately 10 μg of each isocitrate dehydrogenase sample was applied to the gel, and electrophoresis was conducted at pH 8.8, as described in Materials and Methods.

Figure 3.

pH dependence of V_max for the wild-type and alanine-substituted mutant enzymes. WT (•), S95A (○), T78A (▴), and N97A (▵). The activities were measured in various buffers (sodium acetate buffer at pH 4.9–6; imidazole chloride buffer at pH 6–7; triethanolamine chloride buffer at pH 7–7.8). For the pH-rate profile, the activity was measured using 4 mM Mn²⁺, 8 mM isocitrate, and 2 mM NADP in order to ensure saturation of the enzyme over the entire pH range.

Figure 4.

Circular dichroism spectra of wild-type and mutant enzymes. WT (•), T78A (▪), S95A (□), N97A (♦), T78D (○), S95D (▾), and N97D (▿). The CD was measured at pH 7.7 and room temperature, and the molar ellipticity was calculated as described previously (Grodsky et al. 2000).