| Summary: | The aim of the research was to ascertain the nature of the interactions, if any, between the lipoic acid moiety of pyruvate dehydrogenase multienzyme complex from E. coli and the different protein subunits which make up the enzyme. The lipoic acid residues are bound covalently to ε-amino groups of lysine residues on the lipoate acetyltransferase subunits, which form the structural core of the enzyme. It has been proposed that the lipoyl-lysine acts as a flexible couple between the subunits, transferring substrates from one type of enzyme active site to the next in the sequence. The fully extended hydrocarbon chain of the lipoic acid and of the lysine to which it is attached give a distance of 14Å from the dithiolane ring on the lipoic acid to the polypeptide backbone of the lipoate acetyltransferase. Brown and Perham (1976) developed a method for modifying the lipoic acid specifically, which took advantage of the production of acetyl-dihydrolipoic acid in the presence of pyruvate, thus also producing a free thiol group on the lipoic acid residue. This thiol group is available for reaction with reagents such as maleimides. The dissertation describes the modification of the lipoic acid groups with maleimides containing spin-label and fluorescent probes. It is shown that specific modification can only be achieved after precautions have been taken to reduce non-specific modification of other residues. Surprisingly, the rate of modification of the lipoic acid is found to be faster than the rate of loss of total enzyme activity. Results obtained from electron spin resonance spectroscopy and steady-state fluorescence measurements of energy transfer from the fluorophores used to the FAD prosthetic groups on the lipoamide dehydrogenase subunits are presented. The results can be interpreted as showing interactions between groups attached to the lipoic acid moieties and the active sites of the pyruvate decarboxylase and lipoamide dehydrogenase subunits.
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