Abstract
Pig heart mitochondrial NADP-dependent isocitrate dehydrogenase is the most extensively studied among the mammalian isocitrate dehydrogenases. The crystal structure of Escherichia coli isocitrate dehydrogenase and sequence alignment of porcine with E. coli isocitrate dehydrogenase suggests that the porcine Arg(101), Arg(110), Arg(120), and Arg(133) are candidates for roles in substrate binding. The four arginines were separately mutated to glutamine using a polymerase chain reaction method. Wild type and mutant enzymes were each expressed in E. coli, isolated as maltose binding fusion proteins, then cleaved with thrombin, and purified to yield homogeneous porcine isocitrate dehydrogenase. The R120Q mutant has a specific activity, as well as K(m) values for isocitrate, Mn(2+), and NADP(+) similar to wild type enzyme, indicating that Arg(120) is not needed for function. The specific activities of R101Q, R110Q, and R133Q are 1.73, 1.30, and 19.7 micromols/min/mg, respectively, as compared with 39.6 units/mg for wild type enzyme. The R110Q and R133Q enzymes exhibit K(m) values for isocitrate that are increased more than 400- and 165-fold, respectively, as compared with wild type. The K(m) values for Mn(2+), but not for NADP(+), are also elevated indicating that binding of the metal-isocitrate complex is impaired in these mutants. It is proposed that the positive charges of Arg(110) and Arg(133) normally strengthen the binding of the negatively charged isocitrate by electrostatic attraction. The R101Q mutant shows smaller, but significant increases in the K(m) values for isocitrate and Mn(2+); however, the marked decrease in k(cat) suggests a role for Arg(101) in catalysis. The V(max) of wild type enzyme depends on the ionized form of an enzymic group of pK 5.5, and this pK(aes) is similar for the R101Q and R120Q enzymes. In contrast, the pK(aes) for R110Q and R133Q enzymes increases to 6.4 and 7.4, respectively, indicating that the positive charges of Arg(110) and Arg(133) normally lower the pK of the nearby catalytic base to facilitate its ionization. These results may be understood in terms of the structure of the porcine NADP-specific isocitrate dehydrogenase generated by the Insight II Modeler Program, based on the x-ray coordinates of the E. coli enzyme.
Highlights
Expression and Purification of Wild Type and Mutant Enzymes—The porcine NADP-dependent isocitrate dehydrogenase, with glutamine substituted for arginine at each of the positions 101, 110, 120, and 133, were generated using expression vector pMALcIDP1 by a megaprimer PCR method (3)
Based on the sequence alignment of the mammalian and E. coli isocitrate dehydrogenases, consideration of the relatively few amino acids, which are conserved in all species, and analysis of the crystal structures of the E. coli enzyme, Arg101, Arg110, Arg120, and Arg133 were chosen for testing by mutagenesis as candidates for interaction with the carboxylates for isocitrate
Neutral glutamine was selected to replace arginine because it lacks the positive charge of the wild type amino acid but is polar and is similar in size to arginine
Summary
Materials—Oligonucleotides were synthesized by the Operon Technologies, Inc. (Alameda, CA). Site-directed Mutagenesis—A 1.2-kbp cDNA-encoding pig heart mitochondrial NADP-specific isocitrate dehydrogenase was previously cloned into a bacterial expression vector pMal-c2 (New England Biolabs), which yields the enzyme as a fusion protein with maltose-binding protein (11). The digest was dialyzed against 0.018 M triethanolamine chloride buffer, pH 7.1, containing 10% glycerol, 0.06 M Na2SO4, and 1 mM MnSO4 (Buffer B) and applied to a Matrex gel Red-A resin equilibrated with Buffer B to separate pig heart isocitrate dehydrogenase from the maltose-binding protein and thrombin. Wild type and mutant enzymes (0.1–1.0 mg) in 0.4 ml were applied to the column, which was previously equilibrated with 0.1 M triethanolamine chloride buffer, pH 7.7, containing 10% glycerol and 0.3 M Na2SO4. Wild type and mutant proteins (0.15 mg/ml) in 0.025 M triethanolamine chloride buffer, pH 7.7, containing 10% glycerol and 0.075 M Na2SO4 were used for circular dichroism measurements. Out of the models generated from different alignments, a model from the best alignment was chosen by comparing the lowest F values
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