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Ligands of the Mn2+ bound to porcine mitochondrial NADP-dependent isocitrate dehydrogenase, as assessed by mutagenesis.
Biochemistry. 2004 Mar 16; 43(10):2821-8.B

Abstract

Pig heart mitochondrial NADP-dependent isocitrate dehydrogenase requires a divalent metal ion for catalysis, and metal-isocitrate is its preferred substrate. On the basis of the crystal structure of the enzyme-Mn(2+)-isocitrate complex, Asp(252), Asp(275), and Asp(279) were selected as targets for site-directed mutagenesis to evaluate the roles of these residues as ligands of the metal ion. The circular dichroism spectra of the purified mutant enzymes are similar to that of wild-type enzyme indicating there are no appreciable conformational changes. The K(m) values for isocitrate and for Mn(2+) are increased in the asparagine and histidine mutants at positions 252 and 275; while for cysteine mutants at the same positions, the K(m)'s are not changed appreciably. Mutants at position 279 exhibit only a small change in K(m) for isocitrate. These results indicate that Asp(252) and Asp(275) are ligands of enzyme-bound Mn(2+)and influence the binding of Mn(2+)-isocitrate. Cysteine is an acceptable substitute for aspartate as a ligand of Mn(2+). The pK(aes)'s of D252C and D275C enzymes are similar to that of the wild-type enzyme (about 5.2), while the pK(aes) of D279C is a little lower (about 4.7). These findings suggest that the V(max)'s of the D252C, D275C, and D279C enzymes depend on the ionizable form of the same group as in wild-type enzyme and neither Asp(252), Asp(275), nor Asp(279) acts as the general base in the enzymatic reaction. For wild-type enzyme, the pK(aes) varies with the metal ion used with Mg(2+) > Cd(2+) > Mn(2+) > Co(2+), similar to the order of the pK's for these four metal-bound waters. We therefore attribute the pH dependence of V(max) to the deprotonation of the metal-coordinated hydroxyl group of isocitrate bound to isocitrate dehydrogenase.

Authors+Show Affiliations

Department of Chemistry and Biochemistry, University of Delaware, Newark, Delaware 19176, USA.No affiliation info availableNo affiliation info availableNo affiliation info available

Pub Type(s)

Journal Article
Research Support, U.S. Gov't, P.H.S.

Language

eng

PubMed ID

15005617

Citation

Huang, Yu Chu, et al. "Ligands of the Mn2+ Bound to Porcine Mitochondrial NADP-dependent Isocitrate Dehydrogenase, as Assessed By Mutagenesis." Biochemistry, vol. 43, no. 10, 2004, pp. 2821-8.
Huang YC, Grodsky NB, Kim TK, et al. Ligands of the Mn2+ bound to porcine mitochondrial NADP-dependent isocitrate dehydrogenase, as assessed by mutagenesis. Biochemistry. 2004;43(10):2821-8.
Huang, Y. C., Grodsky, N. B., Kim, T. K., & Colman, R. F. (2004). Ligands of the Mn2+ bound to porcine mitochondrial NADP-dependent isocitrate dehydrogenase, as assessed by mutagenesis. Biochemistry, 43(10), 2821-8.
Huang YC, et al. Ligands of the Mn2+ Bound to Porcine Mitochondrial NADP-dependent Isocitrate Dehydrogenase, as Assessed By Mutagenesis. Biochemistry. 2004 Mar 16;43(10):2821-8. PubMed PMID: 15005617.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Ligands of the Mn2+ bound to porcine mitochondrial NADP-dependent isocitrate dehydrogenase, as assessed by mutagenesis. AU - Huang,Yu Chu, AU - Grodsky,Neil B, AU - Kim,Tae-Kang, AU - Colman,Roberta F, PY - 2004/3/10/pubmed PY - 2004/7/13/medline PY - 2004/3/10/entrez SP - 2821 EP - 8 JF - Biochemistry JO - Biochemistry VL - 43 IS - 10 N2 - Pig heart mitochondrial NADP-dependent isocitrate dehydrogenase requires a divalent metal ion for catalysis, and metal-isocitrate is its preferred substrate. On the basis of the crystal structure of the enzyme-Mn(2+)-isocitrate complex, Asp(252), Asp(275), and Asp(279) were selected as targets for site-directed mutagenesis to evaluate the roles of these residues as ligands of the metal ion. The circular dichroism spectra of the purified mutant enzymes are similar to that of wild-type enzyme indicating there are no appreciable conformational changes. The K(m) values for isocitrate and for Mn(2+) are increased in the asparagine and histidine mutants at positions 252 and 275; while for cysteine mutants at the same positions, the K(m)'s are not changed appreciably. Mutants at position 279 exhibit only a small change in K(m) for isocitrate. These results indicate that Asp(252) and Asp(275) are ligands of enzyme-bound Mn(2+)and influence the binding of Mn(2+)-isocitrate. Cysteine is an acceptable substitute for aspartate as a ligand of Mn(2+). The pK(aes)'s of D252C and D275C enzymes are similar to that of the wild-type enzyme (about 5.2), while the pK(aes) of D279C is a little lower (about 4.7). These findings suggest that the V(max)'s of the D252C, D275C, and D279C enzymes depend on the ionizable form of the same group as in wild-type enzyme and neither Asp(252), Asp(275), nor Asp(279) acts as the general base in the enzymatic reaction. For wild-type enzyme, the pK(aes) varies with the metal ion used with Mg(2+) > Cd(2+) > Mn(2+) > Co(2+), similar to the order of the pK's for these four metal-bound waters. We therefore attribute the pH dependence of V(max) to the deprotonation of the metal-coordinated hydroxyl group of isocitrate bound to isocitrate dehydrogenase. SN - 0006-2960 UR - https://www.unboundmedicine.com/medline/citation/15005617/Ligands_of_the_Mn2+_bound_to_porcine_mitochondrial_NADP_dependent_isocitrate_dehydrogenase_as_assessed_by_mutagenesis_ L2 - https://doi.org/10.1021/bi030253f DB - PRIME DP - Unbound Medicine ER -