Mobility of polypeptide chain in the pyruvate dehydrogenase complex revealed by proton NMR

Abstract

Recent studies of several small proteins by NMR spectroscopy and X-ray crystallography have clearly demonstrated significant internal mobility in their structures (see, for example, refs 1–9), which can involve not only amino acid side chains but also larger regions of polypeptide chain. Occasionally a plausible function for this mobility has been suggested^1,9, but there has been no conclusive evidence for a direct connection between intramolecular mobility and a defined step in an enzymatic mechanism. The pyruvate dehydrogenase (PDH) multienzyme complex of Escherichia coli (molecular weight (M_r) 4.5–6 ×10⁶) is one of the largest well defined assemblies of proteins known, comprising multiple copies of three different enzymes^10,11. The substrate is carried in thioester linkage by lipoyl–lysine residues of the lipoate acetyltransferase component, the structural core of the complex. The lipoyl–lysine residues act as swinging arms, carrying substrate between the catalytic centres of the three enzymes^12–15 and between lipoic acid residues attached to different subunits in the lipoate acetyltransferase core^16–18. It has been conjectured that the lipoic acid-containing regions of polypeptide chain might be flexible^19,20 and therefore able to increase greatly the effective radius of a swinging arm¹⁹. We report here unexpectedly sharp lines in the 270-MHz proton NMR spectrum of the enzyme complex that are attributed to remarkable conformational mobility of large regions of polypeptide chain carrying the lipoic acid residues. This mobility would enhance the functional connection of active sites in a multisubunit structure.