Use of dimethyl suberimidate and novel periodate-cleavable bis(imido esters) to study the quaternary structure of the pyruvate dehydrogenase multienzyme complex of Escherichia coli.

Two new symmetrical bis(imido esters), N,N'-bis(2-carboximidoethyl)tartarimide dimethyl ester dihydrochloride and N,N'-bis(2-carboximidomethyl)tartarimide dimethyl ester dihydrochloride, have been synthesized. Tests with the tetrameric enzyme, fructose diphosphate aldolase, show that these reagents closely resemble dimethyl suberimidate in their ability to cross-link protein subunits. However, identification of the cross-linked species, separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis, is greatly facilitated since the cross-links can be broken by a simple treatment with sodium periodate. The periodate cleavage step can be introduced between the two dimensions of a diagonal gel electrophoretic separation, the contributors to a cross-linked species then moving off the diagonal formed by uncross-linked proteins and reverting to the positions in the gel that correspond with their regenerated monomeric form. When the pyruvate dehydrogenase multienzyme complex of Escherichia coli was treated with dimethyl suberimidate or N,N'-bis(2-carboximidoethyl)tartarimide dimethyl ester dihydrochloride, cross-links rapidly formed between the subunits of the transacetylase and lipoamide dehydrogenase components. On the other hand, cross-links failed to form between the subunits of the decarboxylase component themselves, or between the decarboxylase and the other two types of subunit in the complex. Cross-linking experiments with the isolated lipoamide dehydrogenase were compatible with the accepted dimeric structure of this enzyme is free solution, whereas the isolated pyruvate decarboxylase component also failed to cross-link when treated with dimethyl suberimidate in free solution. The cross-linking experiments with the intact multienzyme complex provide evidence for the existence of the lipoamide dehydrogenase dimer in the assembled enzyme and show the need to interpret such experiments with care since, from other evidence, the pyruvate decarboxylase component is known to be bound to the transacetylase "core" of the complex.