Expression and characterization of branched-chain α-ketoacid dehydrogenase kinase from the rat. Is it a histidine-protein kinase?

James R. Davie, R. Max Wynn, Menghsiao Meng, Yi Shuian Huang, Gordon Aalund, David T. Chuang, Kim S. Lau

Research output: Contribution to journalArticle

49 Scopus citations

Abstract

The recombinant rat branched-chain α-ketoacid dehydrogenase kinase has been amplified from rat kidney cDNA, based on the previously reported rat cDNA sequence (Popov, K. M., Zhao, Y., Shimomura, Y., Kuntz, M. J., and Harris, R. A. (1992) J. Biol. Chem. 267, 1312713130). This kinase was expressed in Escherichia coli as a fusion protein with bacterial maltose- binding protein (MBP). Expression was improved by overexpression of chaperonins GroEL and GroES. The MBP-kinase, when reconstituted with lipoylated recombinant E2 (dihydrolipoyl transacylase), catalyzed phosphorylation of recombinant E1 (branched-chain α-ketoacid decarboxylase) with a k(cat) of 20.5 nmol of phosphate/min/nmol of MBP-kinase at 25 °C. Recombinant MBP-kinase alone demonstrated a slow rate of autophosphorylation with a k(cat) of 3.25 pmol of phosphate/min/nmol of kinase at 25 °C. Serine 22 of the kinase was identified as the possible site of autophosphorylation by Edman microsequencing analysis. Autophosphorylated kinase cannot transfer phosphate to E1, indicating that autophosphorylation of kinase is not an intermediate in ATP-dependent phosphorylation of E1. Therefore, despite the reported sequence similarity to prokaryotic histidine protein kinases, the mitochondrial rat branched-chain α-ketoacid dehydrogenase kinase apparently does not phosphorylate E1 via a histidine-mediated phosphotransfer reaction. Significant corrections to the published cDNA sequence of rat branched-chain α-ketoacid dehydrogenase kinase are included.

Original languageEnglish (US)
Pages (from-to)19861-19867
Number of pages7
JournalJournal of Biological Chemistry
Volume270
Issue number34
DOIs
StatePublished - Jan 1 1995

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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