The Subunit Structure of the Escherichia coli 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 80000 (f 5 O/J. 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-(Va1,Leu)-Met-CO,H per 80000 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 80000.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 T I of E . coli K-12, the 80000 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 56 000.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 goo/,, pure, within one week.The pyruvate dehydrogenase complex catalyzes the overall reaction : Pyruvate + NAD+ + CoA + Acetyl-CoA + CO, + NADH + H+. (1) The complex consists of three enzyme components, pyruvate dehydrogenase (El), dihydrolipoamide transacetylase (E2), and dihydrolipoamide dehydrogenase (E3). These components participate in reaction (1) by the following sequence of reactions [1,21: .____

The stoichiometry of polypeptide chains in the pyruvate dehydrogenase multienzyme complex of E. Coli determined by a simple novel method

“…The specific radioactivity of the reagent therefore need not be determined. [6,16,171. No differences in amino acid compositions of components from different strains have been detected in this laboratory.…”

A convenient method of preparation of high-activity urease from Canavalia ensiformis by covalent chromatography and an investigation of its thiol groups with 2,2'-dipyridyl disulphide as a thiol titrant and reactivity probe

“…Bio-Gel A-50m chromatography has been used to separate proteins in the mol.wt. range 100000-several millions (Vogel et al, 1971). …”

The Subunit Structure of the Pyruvate‐Dehydrogenase Complex from Escherichia coli K‐12

“…The procedure involving chromatography on Biogel A-50-M and subsequently on calcium phosphate gel has been described previously [4]. For several experiments we have also purified the enzyme complex by chromatography of crude extracts on calcium phosphate gel alone.…”

“…For several experiments we have also purified the enzyme complex by chromatography of crude extracts on calcium phosphate gel alone. For this purpose crude extracts were prepared as described recently [4]. The extract was diluted with 0.01 M .potassium phosphate buffer pH 7 to a protein concentration of 5mg/nil (usually about 10-fold).…”

“…Our studies on the polypeptide chain composition of the complex [3,4] have shown that the pyruvate dehydrogenase component is a dimer consisting of identical chains with a molecular weight of about 100000, and that the dihydrolipoamide transacetylase consists of identical chains with a molecular weight of about 80000. We have substantiated that the dihydrolipoamide dehydrogenase consists of polypeptide chains with a molecular weight of about 56000 [4-61, and we have now also shown that these chains are identical (0.…”