Specificity determinants for the pyruvate dehydrogenase component reactionmapped with mutated and prosthetic group modified lipoyl domains

Efficient catalysis in the second step of the pyruvate dehydrogenase (E1) c omponent reaction requires a lipoyl group to be attached to a lipoyl domain that displays appropriately positioned specificity residues. As substrates , the human dihydrolipoyl acetyltransferase provides an N-terminal (L1) and an inner (L2) lipoyl domain. We evaluated the specificity requirements for the E1 reaction with 27 mutant L2 (including four substitutions for the li poylated lysine, Lys(173)), with three analogs substituted for the lipoyl g roup on Lys(173), and with selected ii mutants. Besides Lys(173) mutants, o nly E170Q mutation prevented lipoylation. Based on analysis of the structur al stability of mutants by differential scanning calorimetry, alanine subst itutions of residues with aromatic side chains in terminal regions outside the folded portion of the L2 domain significantly decreased the stability o f mutant L2, suggesting specific interactions of these terminal regions wit h the folded domain. E1 reaction rates were markedly reduced by the followi ng substitutions in the L2 domain (equivalent site-L1): L140A, S141A (S14A- L1), T143A, E162A, D172N, and E179A (E52A-L1), These mutants gave diverse c hanges in kinetic parameters. These residues are spread over >24 Angstrom o n one side of the L2 structure, supporting extensive contact between E1 and L2 domain. Alignment of over 40 lipoyl domain sequences supports Ser(141), Thr(143), and Glu(179) serving as specificity residues for use by E1 from eukaryotic sources. Extensive interactions of the lipoyl-lysine prosthetic group within the active site are supported by the limited inhibition of E1 acetylation of native L2 by L2 domains altered either by mutation of Lys(17 3) Or enzymatic addition of lipoate analogs to Lys(173). Thus, efficient us e by mammalian E1 of cognate lipoyl domains derives from unique surface res idues with critical interactions contributed by the universal lipoyl-lysine prosthetic group, key specificity residues, and some conserved residues, p articularly Asp(172) adjacent to Lys(173).