Recognition of the lipoyl domain is the ultimate determinant of substrate channelling in the pyruvate dehydrogenase multienzyme complex

Reductive acetylation of the lipoyl domain (E2plip) of the dihydrolipoyl ac etyltransferase component of the pyruvate dehydrogenase multienzyme complex of Escherichia coli is catalysed specifically by its partner pyruvate deca rboxylase (E1p), and no productive interaction occurs with the analogous 2- oxoglutarate decarboxylase (E1o) of the 2-oxoglutarate dehydrogenase comple x. Residues in the lipoyl-lysine beta -turn region of the unlipoylated E2pl ip domain (E2plip(apo)) undergo significant changes in both chemical shift and transverse relaxation time (T-2) in the presence of E1p but not E1o. Re sidue Gly11, in a prominent surface loop between beta -strands 1 and 2 in t he E2plip domain, was also observed to undergo a significant change in chem ical shift. Addition of pyruvate to the mixture of E2plip(apo) and E1p caus ed larger changes in chemical shift and the appearance of multiple cross-pe aks for certain residues, suggesting that the domain was experiencing more than one type of interaction. Residues in both beta -strands 4 and 5, toget her with those in the prominent surface loop and the following beta -strand 2, appeared to be interacting with E1p, as did a small patch of residues c entred around Glu31. The values of T-2 across the polypeptide chain backbon e were also lower than in the presence of E1p alone, suggesting that E2plip (apo) binds more tightly after the addition of pyruvate. The lipoylated dom ain (E2plip(holo)) also exhibited significant changes in chemical shift and decreases in the overall T-2 relaxation times in the presence of E1p, the residues principally affected being restricted to the half of the domain th at contains the lipoyl-lysine (Lys41) residue. In addition, small chemical shift changes and a general drop in T-2 times in the presence of E1o were o bserved, indicating that E2plip(holo) can interact, weakly but non-producti vely, with E1o. It is evident that recognition of the protein domain is the ultimate determinant of whether reductive acetylation of the lipoyl group occurs, and that this is ensured by a mosaic of interactions with the E1p. (C) 2001 Academic Press.