LIMITED PROTEOLYSIS OF THE PYRUVATE-DEHYDROGENASE MULTI-ENZYME COMPLEX OF ESCHERICHIA-COLI

Incubation of the pyruvate dehydrogenase multienzyme complex of Escherichia coli with trypsin at pH 7.0 leads to limited proteolysis of the constituent polypeptide chains and, more slowly, to loss of enzymic activity. The core component, lipoate acetyltransferase, is the most susceptible to tryptic cleavage, whereas the lipoamide dehydrogenase component is apparently unaffected. The susceptiable peptide bonds in lipoate acetyltransferase involve lysine residues, probably on exposed loops of polypeptide chain. No protection against the action of trypsin is afforded to the individual enzymes by their self‐assembly into the complex, suggesting that no gross conformational changes in the subunits take place on assembly. The principal large fragments of lipoate acetyltransferase chains produced by limited tryptic digestion of native complex have molecular weights (estimated by sodium dodecylsulphate/polyacrylamide gel electrophoresis) of about 39000 and 36000. The fragment of molecular weight 36000 appears to be the ultimate main product. This fragment, presumably a folding domain of some kind, may be represented twice in the lipoate acetyltransferase chain (assumed molecular weight 83000). The major product of tryptic digestion of the native complex remains of high molecular weight. Similarly, the proteolysed lipoate acetyltransferase/lipoamide dehydrogenase subcomplex retains its ability to form a complex of high molecular weight with pyruvate decarboxylase, the third component enzyme. The large folding domains of the lipoate acetyltransferase chains appear to be of major importance in the maintenance of subunit interactions in the enzyme complex. These results with trypsin provide a satisfying rationale for the degradation of the native complex by endogenous proteinases encountered in earlier work. Copyright © 1979, Wiley Blackwell. All rights reserved