The Subunit Structure of the <i>Escherichia coli</i> K‐12 Pyruvate Dehydrogenase Complex

Abstract

The dihydrolipoamide transacetylase component of the Escherichia coli K‐12 pyruvate dehydrogenase complex has been shown to consist of identical or very nearly identical polypeptide chains with a molecular weight of 80 000 (± 5%). Amino acid and tryptic peptide analyses gave a minimum chemical molecular weight of about 80 000. Carboxypeptidase degradation revealed one C‐terminal sequence …Arg‐Arg‐(Val,Leu)‐Met‐CO 2 H per 80 000 molecular weight protein. An N‐terminal amino acid was not found. Dodecylsulfate polyacrylamide gel electrophoresis of the transacetylase subunit (either native, performic acid oxidized, or carboxymethylated) showed one protein band moving as a species with molecular weight of 80 000. These data are at variance with the results of Reed and associates who had found that the transacetylase component of the E. coli Crookes pyruvate dehydrogenase complex consists of identical 36000 molecular weight polypeptide chains. It has been found that in strain W945 T1 of E. coli K‐12, the 80 000 molecular weight transacetylase subunit is cleaved during the purification procedure, yielding a major fragment with a molecular weight of about 38000. It is possible that this cleavage explains the inconsistency between the transacetylase subunit molecular weights. It has been confirmed that the dihydrolipoamide dehydrogenase consists of subunits with molecular weight 56000. A new, relatively rapid purification procedure for the pyruvate dehydrogenase complex is described; it allows the preparation of 200–300 mg enzyme complex, more than 90% pure, within one week.