OVEREXPRESSION AND CHARACTERIZATION OF HUMAN TETRAMERIC PYRUVATE-DEHYDROGENASE AND ITS INDIVIDUAL SUBUNITS

Pyruvate dehydrogenase (E1), an alpha(2) beta(2) tetramer, is the firs t component of the pyruvate dehydrogenase complex which catalyzes a tw o-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 i nto a plasmid pQE-9 and expressed in Escherichia coli M15. A polyhisti dine extension was added at the NH2-termini of the recombinant E1 alph a and E1 beta for the rapid purification of the proteins by Ni-nitrilo triacetic-agarose chromatography, The polyhistidine extension on eithe r E1 alpha or E1 beta subunit did not affect the activity of the recom binant tetrameric E1. Highly purified recombinant human E1 catalyzed t he partial reactions of the oxidative and nonoxidative conversion of p yruvic acid with the same efficiency as E1 purified from bovine kidney , Recombinant human E1 interacted with thiamin pyrophosphate by formin g a charge transfer complex band at 330 nm that changed during the cat alytic cycle, Recombinant human E1 was phosphorylated by E1-kinase (wi th concomitant inactivation) by incorporating nearly three phosphoryl groups per mole of E1. When expressed individually, E1 alpha and E1 be ta subunits lacked any catalytic activity in the oxidative or nonoxida tive reactions, Spectral studies demonstrated that there was no thiami n pyrophosphate binding to either recombinant E1 alpha or E1 beta subu nit, The E1 alpha subunit retained the ability to be phosphorylated; h owever, the incorporation of phosphoryl groups into recombinant E1 alp ha alone was only about 12% of that observed with the tetrameric E1. T hese findings show that both subunits are required for formation of th e active center and catalysis. (C) 1995 Academic Press, Inc.