Bovine Kidney Pyruvate Dehydrogenase Complex

Abstract

Bovine kidney pyruvate dehydrogenase multienzyme complex is inactivated rapidly by papain. However, none of the component activities of the complex is destroyed during inactivation of the overall reaction. The core component, lipoate acetyltransferase, is cleaved by papain to give principal fragments with Mr 26500 and 26000 (as determined by dodecylsulfate gel electrophoresis). Much more slowly, the α chain of the pyruvate dehydrogenase component is attacked. Fragmented lipoate acetyltransferase retains its complete enzymatic activity and remains of high molecular weight. It is unable, however, to bind the other component enzymes, pyruvate dehydrogenase and lipoamide dehydrogenase. Therefore, the multienzyme complex is disassembled when treated with papain. A method is described which allows the rapid and convenient isolation of nicked lipoate acetyltransferase as well as unfragmented pyruvate dehydrogenase and lipoamide dehydrogenase from papain‐treated complex under very mild conditions. The two uncleaved component enzymes have identical properties and similar specific activities as enzyme preparations obtained by other, more laborious procedures.

Conformational mobility of polypeptide chains in the 2‐oxo acid dehydrogenase complexes from ox heart revealed by proton NMR spectroscopy

“…For that complex we were able, by limited proteolysis experiments, to identify the highly mobile regions as being associated with the lipoic acid-containing regions of the lipoate acetyltransferase (E2) polypeptide chains. Large lipoic acidcontaining regions of the pyruvate dehydrogenase complex from ox kidney [12] and ox heart [13] can be released by limited proteolysis and appear to protrude from the E2 core. The same is probably true of the 2-oxoglutarate dehydrogenase complex from ox kidney [20].…”

“…Studies of the pyruvate dehydrogenase complexes from E. coli [8][9][10], from B. stearothermophilus [ 11 ] and from ox kidney [ 12] and ox heart [ 13] by limited proteolysis and electron microscopy suggested that lipoic acid-containing regions of the E2 chains protrude from the E2 core between the E1 and E3 subunits. An extraordinary degree of conformational mobility of large segments of the E2 chains encompassing the lipoyl-lysine residues was demonstrated by proton NMR spectroscopy of the E. coli complex [ 14].…”

Limited proteolysis of 2‐oxoglutarate dehydrogenase multienzyme complex from bovine kidney

“…In contrast, during treatment with trypsin or bromelain, the El band was cleaved faster than E2. Just as with bovine kidney PDC [3,4,7] OGDC was disassembled into its enzyme components during treatment with papain or elastase. This is demonstrated in fig.3 showing the results of sucrose density gradient centrifugation of papain-treated OGDC.…”

“…The experimental conditions for protease treatment of OGDC were identical as described for PDC in [3,7].…”

Cloning and sequence analysis of the genes encoding the dihydrolipoamide acetyltransferase and dihydrolipoamide dehydrogenase components of the pyruvate dehydrogenase multienzyme complex of Bacillus stearothermophilus

“…The M, of the individual components as well as the morphology of the E2 core [3, 41 closely resemble those of the PDH complexes of eukaryotes. Its thermostability makes the B. stearothermophilus PDH complex a good system for studying the structure of a 2-0x0-acid dehydrogenase complex based on icosahedral symmetry, which differs from the PDH and 2-oxoglutarate dehydrogenase complexes of Escherichia coli in that the latter have octahedral E2 cores [5, 61.…”