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Mutation of the predicted ATP binding site inactivates both activities of isocitrate dehydrogenase kinase/phosphatase.
J Biol Chem. 1989 Aug 15; 264(23):13775-9.JB

Abstract

In Escherichia coli, the reversible phosphorylation of isocitrate dehydrogenase (IDH) is catalyzed by a bifunctional protein: IDH kinase/phosphatase. Although both IDH kinase and IDH phosphatase require ATP, the amino acid sequence of IDH kinase/phosphatase contains a single sequence that matches the consensus for ATP binding sites. A mutation that converted the "invariant" lysine (residue 336) of this consensus sequence to a methionine reduced the activities of both IDH kinase and IDH phosphatase by factors of greater than 500, to levels below the detection limits of the assays. The apparent elimination of both IDH kinase and IDH phosphatase by this mutation is consistent with the proposal that these activities share a common ATP binding site and that these reactions may occur at the same active site. Although conversion of Lys336 to a methionine eliminated detectable IDH kinase activity as measured in vitro, the mutant allele retained the ability to complement an aceK deletion mutation, restoring the ability of these cells to grow on minimal acetate medium. Complementation apparently resulted because the mutant protein retained sufficient activity to phosphorylate IDH in vivo. To determine whether the enzymatic assays performed in vitro had correctly reflected the activity of the mutant protein in vivo, we measured the rates at which mutant and wild-type cultures could incorporate [32P]inorganic phosphate into IDH. The wild-type culture achieved maximal incorporation in less than 3 min. In contrast, 32P incorporation was only barely detectable after 30 min in the mutant culture, indicating that the activity of the mutant protein is, indeed, greatly reduced in vivo. The ability of the mutant allele to complement an aceK null mutation thus suggests that IDH kinase/phosphatase levels in wild-type cells are in great excess over what is required for steady-state growth on acetate medium.

Authors+Show Affiliations

Department of Biochemistry, University of Minnesota, Minneapolis 55455.No affiliation info availableNo affiliation info available

Pub Type(s)

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

Language

eng

PubMed ID

2547774

Citation

Stueland, C S., et al. "Mutation of the Predicted ATP Binding Site Inactivates Both Activities of Isocitrate Dehydrogenase Kinase/phosphatase." The Journal of Biological Chemistry, vol. 264, no. 23, 1989, pp. 13775-9.
Stueland CS, Ikeda TP, LaPorte DC. Mutation of the predicted ATP binding site inactivates both activities of isocitrate dehydrogenase kinase/phosphatase. J Biol Chem. 1989;264(23):13775-9.
Stueland, C. S., Ikeda, T. P., & LaPorte, D. C. (1989). Mutation of the predicted ATP binding site inactivates both activities of isocitrate dehydrogenase kinase/phosphatase. The Journal of Biological Chemistry, 264(23), 13775-9.
Stueland CS, Ikeda TP, LaPorte DC. Mutation of the Predicted ATP Binding Site Inactivates Both Activities of Isocitrate Dehydrogenase Kinase/phosphatase. J Biol Chem. 1989 Aug 15;264(23):13775-9. PubMed PMID: 2547774.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Mutation of the predicted ATP binding site inactivates both activities of isocitrate dehydrogenase kinase/phosphatase. AU - Stueland,C S, AU - Ikeda,T P, AU - LaPorte,D C, PY - 1989/8/15/pubmed PY - 1989/8/15/medline PY - 1989/8/15/entrez SP - 13775 EP - 9 JF - The Journal of biological chemistry JO - J Biol Chem VL - 264 IS - 23 N2 - In Escherichia coli, the reversible phosphorylation of isocitrate dehydrogenase (IDH) is catalyzed by a bifunctional protein: IDH kinase/phosphatase. Although both IDH kinase and IDH phosphatase require ATP, the amino acid sequence of IDH kinase/phosphatase contains a single sequence that matches the consensus for ATP binding sites. A mutation that converted the "invariant" lysine (residue 336) of this consensus sequence to a methionine reduced the activities of both IDH kinase and IDH phosphatase by factors of greater than 500, to levels below the detection limits of the assays. The apparent elimination of both IDH kinase and IDH phosphatase by this mutation is consistent with the proposal that these activities share a common ATP binding site and that these reactions may occur at the same active site. Although conversion of Lys336 to a methionine eliminated detectable IDH kinase activity as measured in vitro, the mutant allele retained the ability to complement an aceK deletion mutation, restoring the ability of these cells to grow on minimal acetate medium. Complementation apparently resulted because the mutant protein retained sufficient activity to phosphorylate IDH in vivo. To determine whether the enzymatic assays performed in vitro had correctly reflected the activity of the mutant protein in vivo, we measured the rates at which mutant and wild-type cultures could incorporate [32P]inorganic phosphate into IDH. The wild-type culture achieved maximal incorporation in less than 3 min. In contrast, 32P incorporation was only barely detectable after 30 min in the mutant culture, indicating that the activity of the mutant protein is, indeed, greatly reduced in vivo. The ability of the mutant allele to complement an aceK null mutation thus suggests that IDH kinase/phosphatase levels in wild-type cells are in great excess over what is required for steady-state growth on acetate medium. SN - 0021-9258 UR - https://www.unboundmedicine.com/medline/citation/2547774/Mutation_of_the_predicted_ATP_binding_site_inactivates_both_activities_of_isocitrate_dehydrogenase_kinase/phosphatase_ DB - PRIME DP - Unbound Medicine ER -