Use of trypsin and lipoamidase to study the role of lipoic acid moieties in the pyruvate and .alpha.-ketoglutarate dehydrogenase complexes of Escherichia coli

Abstract

The relationships between release of 3H-labeled lipoyl moieties by trypsin and lipoamidase and accompanying loss of overall enzymatic activity of the E. coli pyruvate and .alpha.-ketoglutarate dehydrogenase complexes were studied. Trypsin releases lipoyl domains together with their covalently attached lipoyl moieties from the inner core of the dihydro lipoyl transacetylase and the dihydrolipoyl transuccinylase whereas lipoamidase releases only the lipiyl moieties. Apparently, the release of lipoyl domains by trypsin and release of lipoyl moieties by lipoamidase proceeded at faster rates than the accompanying loss of overall activity of the 2 complexes. Trypsin relased about 1/2 of the lipoyl domains in the pyruvate dehydrogenase complex without significant effect on the overall activity. A model is presented to explain these and other observations on active-site coupling via lipoyl moieties.