Expression and characterization of branched-chain alpha-ketoacid dehydrogenase kinase from the rat. Is it a histidine-protein kinase?
The recombinant rat branched-chain alpha-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, 13127-13130). 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 alpha-ketoacid decarboxylase) with a kcat of 28.5 nmol of phosphate/min/nmol of MBP-kinase at 25 degrees C. Recombinant MBP-kinase alone demonstrated a slow rate of autophosphorylation with a kcat of 3.25 pmol of phosphate/min/nmol of kinase at 25 degrees 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 alpha-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 alpha-ketoacid dehydrogenase kinase are included.
Davie
J R
JR
Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas 75235, USA.
Wynn
R M
RM
Meng
M
M
Huang
Y S
YS
Aalund
G
G
Chuang
D T
DT
Lau
K S
KS
D018528:ATP-Binding Cassette Transporters; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002352:Carrier Proteins / Q000235:genetics; D017931:DNA Primers / Q000235:genetics; D018076:DNA, Complementary / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D029968:Escherichia coli Proteins; D015870:Gene Expression; D000071677:Histidine Kinase; D007668:Kidney / Q000201:enzymology; D007700:Kinetics; D058619:Maltose-Binding Proteins; D008928:Mitochondria / Q000201:enzymology; D008969:Molecular Sequence Data; D009004:Monosaccharide Transport Proteins; D010766:Phosphorylation; D011494:Protein Kinases / Q000235:genetics* / Q000378:metabolism; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000235:genetics
D018528:ATP-Binding Cassette Transporters; D002352:Carrier Proteins; D017931:DNA Primers; D018076:DNA, Complementary; D029968:Escherichia coli Proteins; D058619:Maltose-Binding Proteins; D009004:Monosaccharide Transport Proteins; D011993:Recombinant Fusion Proteins; C461599:maltose transport system, E coli; D011494:Protein Kinases; C035627:(3-methyl-2-oxobutanoate dehydrogenase (lipoamide)) kinase; D000071677:Histidine Kinase