Overexpression and characterization of human tetrameric pyruvate dehydrogenase and its individual subunits.
Pyruvate dehydrogenase (E1), an alpha 2 beta 2 tetramer, is the first component of the pyruvate dehydrogenase complex which catalyzes a two-step oxidative decarboxylation of pyruvic acid. To overexpress human E1 and its subunits individually, cDNAs for the mature forms of human E1 alpha and E1 beta were subcloned either individually or together into a plasmid pQE-9 and expressed in Escherichia coli M15. A polyhistidine extension was added at the NH2-termini of the recombinant E1 alpha and E1 beta for the rapid purification of the proteins by Ni-nitrilotriacetic-agarose chromatography. The polyhistidine extension on either E1 alpha or E1 beta subunit did not affect the activity of the recombinant tetrameric E1. Highly purified recombinant human E1 catalyzed the partial reactions of the oxidative and nonoxidative conversion of pyruvic acid with the same efficiency as E1 purified from bovine kidney. Recombinant human E1 interacted with thiamin pyrophosphate by forming a charge transfer complex band at 330 nm that changed during the catalytic cycle. Recombinant human E1 was phosphorylated by E1-kinase (with concomitant inactivation) by incorporating nearly three phosphoryl groups per mole of E1. When expressed individually, E1 alpha and E1 beta subunits lacked any catalytic activity in the oxidative or nonoxidative reactions. Spectral studies demonstrated that there was no thiamin pyrophosphate binding to either recombinant E1 alpha or E1 beta subunit. The E1 alpha subunit retained the ability to be phosphorylated; however, the incorporation of phosphoryl groups into recombinant E1 alpha alone was only about 12% of that observed with the tetrameric E1. These findings show that both subunits are required for formation of the active center and catalysis.
Korotchkina
L G
LG
Department of Biochemistry, Case Western Reserve University School of Medicine, Cleveland, Ohio 44106, USA.
Tucker
M M
MM
Thekkumkara
T J
TJ
Madhusudhan
K T
KT
Pons
G
G
Kim
H
H
Patel
M S
MS
D001483:Base Sequence; D003001:Cloning, Molecular; D017931:DNA Primers; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D008969:Molecular Sequence Data; D010766:Phosphorylation; D010770:Phosphotransferases / Q000494:pharmacology; D011487:Protein Conformation; D011768:Pyruvate Dehydrogenase Complex / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000235:genetics; D013835:Thiamine Pyrophosphate / Q000378:metabolism
D017931:DNA Primers; D011768:Pyruvate Dehydrogenase Complex; D011994:Recombinant Proteins; D010770:Phosphotransferases; D013835:Thiamine Pyrophosphate