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Identification of Mn2+-binding aspartates from alpha, beta, and gamma subunits of human NAD-dependent isocitrate dehydrogenase.
J Biol Chem. 2006 Jul 28; 281(30):21073-81.JB

Abstract

The human NAD-dependent isocitrate dehydrogenase (IDH), with three types of subunits present in the ratio of 2alpha:1beta:1gamma, requires a divalent metal ion to catalyze the oxidative decarboxylation of isocitrate. With the aim of identifying ligands of the enzyme-bound Mn(2+), we mutated aspartates on the alpha, beta, or gamma subunits. Mutagenesis target sites were based on crystal structures of metal-isocitrate complexes of Escherichia coli and pig mitochondrial NADP-IDH and sequence alignments. Aspartates replaced by asparagine or cysteine were 206, 230, and 234 of the alpha subunit and those corresponding to alpha-Asp-206: 217 of the beta subunit and 215 of the gamma subunit. Each expressed, purified mutant enzyme has two wild-type subunits and one subunit with a single mutation. Specific activities of WT, alpha-D206N, alpha-D230C, alpha-D234C, beta-D217N, and gamma-D215N enzymes are 22, 29, 1.4, 0.2, 7.3 and 3.7 micromol of NADH/min/mg, respectively, whereas alpha-D230N and alpha-D234N enzymes showed no activity. The K(m,Mn(2+)) for alpha-D230C and gamma-D215N are increased 32- and 100-fold, respectively, along with elevations in K(m,isocitrate). The K(m,NAD) of alpha-D230C is increased 16-fold, whereas that of beta-D217N is elevated 10-fold. For all the mutants K(m,isocitrate) is decreased by ADP, indicating that these aspartates are not needed for normal ADP activation. This study demonstrates that alpha-Asp-230 and alpha-Asp-234 are critical for catalytic activity, but alpha-Asp-206 is not needed; alpha-Asp-230 and gamma-Asp-215 may interact directly with the Mn(2+); and alpha-Asp-230 and beta-Asp-217 contribute to the affinity of the enzyme for NAD. These results suggest that the active sites of the human NAD-IDH are shared between alpha and gamma subunits and between alpha and beta subunits.

Authors+Show Affiliations

Department of Chemistry and Biochemistry, University of Delaware, Newark, 19716, USA.No affiliation info availableNo affiliation info availableNo affiliation info available

Pub Type(s)

Journal Article
Research Support, N.I.H., Extramural

Language

eng

PubMed ID

16737955

Citation

Soundar, Sambanthamurthy, et al. "Identification of Mn2+-binding Aspartates From Alpha, Beta, and Gamma Subunits of Human NAD-dependent Isocitrate Dehydrogenase." The Journal of Biological Chemistry, vol. 281, no. 30, 2006, pp. 21073-81.
Soundar S, O'hagan M, Fomulu KS, et al. Identification of Mn2+-binding aspartates from alpha, beta, and gamma subunits of human NAD-dependent isocitrate dehydrogenase. J Biol Chem. 2006;281(30):21073-81.
Soundar, S., O'hagan, M., Fomulu, K. S., & Colman, R. F. (2006). Identification of Mn2+-binding aspartates from alpha, beta, and gamma subunits of human NAD-dependent isocitrate dehydrogenase. The Journal of Biological Chemistry, 281(30), 21073-81.
Soundar S, et al. Identification of Mn2+-binding Aspartates From Alpha, Beta, and Gamma Subunits of Human NAD-dependent Isocitrate Dehydrogenase. J Biol Chem. 2006 Jul 28;281(30):21073-81. PubMed PMID: 16737955.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Identification of Mn2+-binding aspartates from alpha, beta, and gamma subunits of human NAD-dependent isocitrate dehydrogenase. AU - Soundar,Sambanthamurthy, AU - O'hagan,Molly, AU - Fomulu,Kenneth S, AU - Colman,Roberta F, Y1 - 2006/05/31/ PY - 2006/6/2/pubmed PY - 2006/9/19/medline PY - 2006/6/2/entrez SP - 21073 EP - 81 JF - The Journal of biological chemistry JO - J. Biol. Chem. VL - 281 IS - 30 N2 - The human NAD-dependent isocitrate dehydrogenase (IDH), with three types of subunits present in the ratio of 2alpha:1beta:1gamma, requires a divalent metal ion to catalyze the oxidative decarboxylation of isocitrate. With the aim of identifying ligands of the enzyme-bound Mn(2+), we mutated aspartates on the alpha, beta, or gamma subunits. Mutagenesis target sites were based on crystal structures of metal-isocitrate complexes of Escherichia coli and pig mitochondrial NADP-IDH and sequence alignments. Aspartates replaced by asparagine or cysteine were 206, 230, and 234 of the alpha subunit and those corresponding to alpha-Asp-206: 217 of the beta subunit and 215 of the gamma subunit. Each expressed, purified mutant enzyme has two wild-type subunits and one subunit with a single mutation. Specific activities of WT, alpha-D206N, alpha-D230C, alpha-D234C, beta-D217N, and gamma-D215N enzymes are 22, 29, 1.4, 0.2, 7.3 and 3.7 micromol of NADH/min/mg, respectively, whereas alpha-D230N and alpha-D234N enzymes showed no activity. The K(m,Mn(2+)) for alpha-D230C and gamma-D215N are increased 32- and 100-fold, respectively, along with elevations in K(m,isocitrate). The K(m,NAD) of alpha-D230C is increased 16-fold, whereas that of beta-D217N is elevated 10-fold. For all the mutants K(m,isocitrate) is decreased by ADP, indicating that these aspartates are not needed for normal ADP activation. This study demonstrates that alpha-Asp-230 and alpha-Asp-234 are critical for catalytic activity, but alpha-Asp-206 is not needed; alpha-Asp-230 and gamma-Asp-215 may interact directly with the Mn(2+); and alpha-Asp-230 and beta-Asp-217 contribute to the affinity of the enzyme for NAD. These results suggest that the active sites of the human NAD-IDH are shared between alpha and gamma subunits and between alpha and beta subunits. SN - 0021-9258 UR - https://www.unboundmedicine.com/medline/citation/16737955/Identification_of_Mn2+_binding_aspartates_from_alpha_beta_and_gamma_subunits_of_human_NAD_dependent_isocitrate_dehydrogenase_ L2 - http://www.jbc.org/cgi/pmidlookup?view=long&pmid=16737955 DB - PRIME DP - Unbound Medicine ER -