BOVINE KIDNEY PYRUVATE-DEHYDROGENASE COMPLEX - LIMITED PROTEOLYSIS AND MOLECULAR-STRUCTURE OF THE LIPOATE ACETYLTRANSFERASE COMPONENT

  • 1 January 1980
    • journal article
    • research article
    • Vol. 112  (3) , 589-599
Abstract
Bovine kidney pyruvate dehydrogenase multienzyme complex is inactivated by elastase in a similar manner as described earlier for papain. The core component, lipoate acetyltransferase, is cleaved by elastase into an active fragment (MW 26,000) and a fragment with apparent MW of 45,000 as analyzed by dodecylsulfate gel electrophoresis. Due to the fragmentation of the core, the enzyme complex is disassembled into its component enzymes which retain their complete enzymatic activities as assayed separately. A different mechanism was found for the inactivation of pyruvate dehydrogenase complex with trypsin and some other proteases (chymotrypsin, clostripain). In these cases, the pyruvate dehydrogenase component is inactivated rapidly by limited proteolysis. More slowly, the enzyme complex is disassembled simultaneously with fragmentation of the lipoate acetyltransferase which again results in an active fragment of MW 26,000 and another fragment of apparent MW 45,000. Upon prolonged proteolysis, the latter fragment is cleaved further to give products of MW 36,000 or lower. The enzyme-bound lipoyl residues of the pyruvate dehydrogenase complex have been labeled covalently by incubation with [2-14C]pyruvate. After treatment of this [14C]acetyl-enzyme with papain, elastase or trypsin, radioactivity was associated exclusively with the 45,000 MW and 36,000 MW fragments but not with the active 26,000 MW fragment. Apparently, the bovine kidney lipoate acetyltransferase core is composed of 60 subunits each consisting of 2 dissimilar folding domains. One of these contains the intersubunit binding sites as well as the active center for transacylation; the other possesses the enzyme-bound lipoyl residues.

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