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

• Published in: Journal of molecular biology Vol. 305; no. 1; pp. 49 - 60
• Main Authors: Jones, D.Dafydd, Stott, Katherine M, Reche, Pedro A, Perham, Richard N
• Format: Journal Article
• Language: English
• Published: England Elsevier Ltd 05.01.2001
• Subjects: Acetylation; Acetyltransferases - chemistry; Acetyltransferases - metabolism; Animals; Apoenzymes - chemistry; Apoenzymes - metabolism; Binding Sites; Carboxy-Lyases - chemistry; Carboxy-Lyases - metabolism; Cattle; Dihydrolipoyllysine-Residue Acetyltransferase; Escherichia coli; Escherichia coli - enzymology; Holoenzymes - chemistry; Holoenzymes - metabolism; lipoyl domain; lipoylation; Models, Molecular; Nuclear Magnetic Resonance, Biomolecular; Protein Binding; Protein Structure, Quaternary; Protein Structure, Secondary; Protein Structure, Tertiary; protein-protein interaction; Pyruvate Decarboxylase - chemistry; Pyruvate Decarboxylase - metabolism; pyruvate dehydrogenase complex; Pyruvate Dehydrogenase Complex - chemistry; Pyruvate Dehydrogenase Complex - metabolism; Pyruvic Acid - metabolism; Serum Albumin - metabolism; Substrate Specificity; E1o; E2p; E2plip; E2plip apo; E1p; T 2; lipoyl domain; BSA; NMR; PDH; E2plip holo; OGDH; pyruvate dehydrogenase complex; protein-protein interaction; BCDH; HSQC; LplA; lipoylation
• ISSN: 0022-2836; 1089-8638
• DOI: 10.1006/jmbi.2000.4257

Reductive acetylation of the lipoyl domain (E2plip) of the dihydrolipoyl acetyltransferase component of the pyruvate dehydrogenase multienzyme complex of Escherichia coli is catalysed specifically by its partner pyruvate decarboxylase (E1p), and no productive interaction occurs with the analogous 2-oxoglutarate decarboxylase (E1o) of the 2-oxoglutarate dehydrogenase complex. Residues in the lipoyl-lysine β-turn region of the unlipoylated E2plip domain (E2plip apo) undergo significant changes in both chemical shift and transverse relaxation time ( T 2) in the presence of E1p but not E1o. Residue Gly11, in a prominent surface loop between β-strands 1 and 2 in the E2plip domain, was also observed to undergo a significant change in chemical shift. Addition of pyruvate to the mixture of E2plip apo and E1p caused larger changes in chemical shift and the appearance of multiple cross-peaks for certain residues, suggesting that the domain was experiencing more than one type of interaction. Residues in both β-strands 4 and 5, together with those in the prominent surface loop and the following β-strand 2, appeared to be interacting with E1p, as did a small patch of residues centred around Glu31. The values of T 2 across the polypeptide chain backbone were also lower than in the presence of E1p alone, suggesting that E2plip apo binds more tightly after the addition of pyruvate. The lipoylated domain (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 that 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 observed, indicating that E2plip holo can interact, weakly but non-productively, 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 Elp.