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Bacillus subtilis isocitrate dehydrogenase. A substrate analogue for Escherichia coli isocitrate dehydrogenase kinase/phosphatase.
J Biol Chem. 2002 Mar 01; 277(9):7567-73.JB

Abstract

In Escherichia coli, the homodimeric Krebs cycle enzyme isocitrate dehydrogenase (EcIDH) is regulated by reversible phosphorylation of a sequestered active site serine. The phosphorylation cycle is catalyzed by a bifunctional protein, IDH kinase/phosphatase (IDH-K/P). To better understand the nature of the interaction between EcIDH and IDH-K/P, we have examined the ability of an IDH homologue from Bacillus subtilis (BsIDH) to serve as a substrate for the kinase and phosphatase activities. BsIDH exhibits extensive sequence and structural similarities with EcIDH, particularly around the phosphorylated serine. Our previous crystallographic analysis revealed that the active site architecture of these two proteins is almost completely conserved. We now expand the comparison to include a number of biochemical properties. Both IDHs display nearly equivalent steady-state kinetic parameters for the dehydrogenase reaction. Both proteins are also phosphorylated by IDH-K/P in the same ratio (1 mole of phosphate per mole of monomer), and this stoichiometric phosphorylation correlates with an equivalent inhibition of IDH activity. Furthermore, tandem electrospray mass spectrometry demonstrates that BsIDH, like EcIDH, is phosphorylated on the corresponding active site serine residue (Ser-104). Despite the high degree of sequence, functional, and structural congruence between these two proteins, BsIDH is surprisingly a much poorer substrate of IDH-K/P than is EcIDH, with Michaelis constants for the kinase and phosphatase activities elevated by 60- and 3,450-fold, respectively. These drastically disparate values might result from restricted access to the active site cavity and/or from the lack of a potential docking site for IDH-K/P.

Authors+Show Affiliations

Department of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota, 321 Church Street SE, Minneapolis, MN 55455, USA.No affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info available

Pub Type(s)

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

Language

eng

PubMed ID

11751849

Citation

Singh, Satinder K., et al. "Bacillus Subtilis Isocitrate Dehydrogenase. a Substrate Analogue for Escherichia Coli Isocitrate Dehydrogenase Kinase/phosphatase." The Journal of Biological Chemistry, vol. 277, no. 9, 2002, pp. 7567-73.
Singh SK, Miller SP, Dean A, et al. Bacillus subtilis isocitrate dehydrogenase. A substrate analogue for Escherichia coli isocitrate dehydrogenase kinase/phosphatase. J Biol Chem. 2002;277(9):7567-73.
Singh, S. K., Miller, S. P., Dean, A., Banaszak, L. J., & LaPorte, D. C. (2002). Bacillus subtilis isocitrate dehydrogenase. A substrate analogue for Escherichia coli isocitrate dehydrogenase kinase/phosphatase. The Journal of Biological Chemistry, 277(9), 7567-73.
Singh SK, et al. Bacillus Subtilis Isocitrate Dehydrogenase. a Substrate Analogue for Escherichia Coli Isocitrate Dehydrogenase Kinase/phosphatase. J Biol Chem. 2002 Mar 1;277(9):7567-73. PubMed PMID: 11751849.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Bacillus subtilis isocitrate dehydrogenase. A substrate analogue for Escherichia coli isocitrate dehydrogenase kinase/phosphatase. AU - Singh,Satinder K, AU - Miller,Stephen P, AU - Dean,Antony, AU - Banaszak,Leonard J, AU - LaPorte,David C, Y1 - 2002/01/07/ PY - 2001/12/26/pubmed PY - 2002/4/2/medline PY - 2001/12/26/entrez SP - 7567 EP - 73 JF - The Journal of biological chemistry JO - J Biol Chem VL - 277 IS - 9 N2 - In Escherichia coli, the homodimeric Krebs cycle enzyme isocitrate dehydrogenase (EcIDH) is regulated by reversible phosphorylation of a sequestered active site serine. The phosphorylation cycle is catalyzed by a bifunctional protein, IDH kinase/phosphatase (IDH-K/P). To better understand the nature of the interaction between EcIDH and IDH-K/P, we have examined the ability of an IDH homologue from Bacillus subtilis (BsIDH) to serve as a substrate for the kinase and phosphatase activities. BsIDH exhibits extensive sequence and structural similarities with EcIDH, particularly around the phosphorylated serine. Our previous crystallographic analysis revealed that the active site architecture of these two proteins is almost completely conserved. We now expand the comparison to include a number of biochemical properties. Both IDHs display nearly equivalent steady-state kinetic parameters for the dehydrogenase reaction. Both proteins are also phosphorylated by IDH-K/P in the same ratio (1 mole of phosphate per mole of monomer), and this stoichiometric phosphorylation correlates with an equivalent inhibition of IDH activity. Furthermore, tandem electrospray mass spectrometry demonstrates that BsIDH, like EcIDH, is phosphorylated on the corresponding active site serine residue (Ser-104). Despite the high degree of sequence, functional, and structural congruence between these two proteins, BsIDH is surprisingly a much poorer substrate of IDH-K/P than is EcIDH, with Michaelis constants for the kinase and phosphatase activities elevated by 60- and 3,450-fold, respectively. These drastically disparate values might result from restricted access to the active site cavity and/or from the lack of a potential docking site for IDH-K/P. SN - 0021-9258 UR - https://www.unboundmedicine.com/medline/citation/11751849/Bacillus_subtilis_isocitrate_dehydrogenase__A_substrate_analogue_for_Escherichia_coli_isocitrate_dehydrogenase_kinase/phosphatase_ L2 - https://linkinghub.elsevier.com/retrieve/pii/S0021-9258(19)82318-9 DB - PRIME DP - Unbound Medicine ER -