Robert M. Oliver

Crystallization of a dihydrolipoyl transacetylase-dihydrolipoyl dehydrogenase subcomplex and its implications regarding the subunit structure of the pyruvate dehydrogenase complex from Escherichia coli

Abstract A subcomplex consisting of dihydrolipoyl transacetylase and dihydrolipoyl dehydrogenase, two of the three enzymes comprising the Escherichia coli pyruvate dehydrogenase complex, has been crystallized. X-ray diffraction data establish that the space group is P 2 1 3 with unit cell dimension a=211 .5 A . The unit cell contains four molecules of the subcomplex, each possessing 3-fold crystallographic and molecular symmetry. This finding, together with biochemical and electron microscopic data reported elsewhere, establish unequivocally that dihydrolipoyl transacetylase, the core enzyme of the pyruvate dehydrogenase complex, consists of 24 identical subunits with octahedral (432) symmetry. In the case presented here, the 432 symmetry of the transacetylase is reduced to 3-fold symmetry in the subcomplex by the addition of dihydrolipoyl dehydrogenase subunits. Crystal density measurements indicate that the dihydrolipoyl transacetylase present in these crystals is considerably smaller than the core mass generally reported for intact transacetylase. The implications of these findings are discussed with respect to the subunit stoichiometry and structure of the E. coli pyruvate dehydrogenase complex.

Structure-Function Relationships in Pyruvate and α-Ketoglutarate Dehydrogenase Complexes

Enzyme systems that catalyze a lipoic acid-mediated oxidative decarboxylation of pyruvate and α-ketoglutarate have been isolated from microbial and eukaryotic cells as functional units with molecular weights in the millions. Each complex consists of three catalytic components: pyruvate dehydrogenase or α -ketoglutarate dehydrogenase (E1); dihydrolipoyl transacetylase or dihydrolipoyl trans-succinylase (E2); and dihydrolipoyl dehydrogenase (E3), a flavopro-tein that is a common component of the two complexes. These three enzymes, acting in sequence, catalyze1 the reactions shown in Fig. 1, E1 catalyzes both the decarboxylation of the α -keto acid (reaction 1) and the subsequent reductive acylation of the lipoyl moiety (reaction 2) that is covalently bound2 to E2. E2 catalyzes the tran-sacylation step (reaction 3), and E3 catalyzes reoxidation of the dihydrolipoyl moiety with NAD+ as the ultimate electron acceptor (reactions 4 and 5). The pyruvate dehydrogenase complexes from eukaryotic cells also contain small amounts of two regulatory enzymes, a kinase and a phosphatase, that modulate the activity of E1 by phosphorylation and dephosphorylation, respectively.3