ABSTRACT
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 beta-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 beta-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 beta-strands 4 and 5, together 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 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.
Subject(s)
Acetyltransferases/chemistry , Acetyltransferases/metabolism , Escherichia coli/enzymology , Pyruvate Dehydrogenase Complex/chemistry , Pyruvate Dehydrogenase Complex/metabolism , Acetylation , Animals , Apoenzymes/chemistry , Apoenzymes/metabolism , Binding Sites , Carboxy-Lyases/chemistry , Carboxy-Lyases/metabolism , Cattle , Dihydrolipoyllysine-Residue Acetyltransferase , Holoenzymes/chemistry , Holoenzymes/metabolism , Models, Molecular , Nuclear Magnetic Resonance, Biomolecular , Protein Binding , Protein Structure, Quaternary , Protein Structure, Secondary , Protein Structure, Tertiary , Pyruvate Decarboxylase/chemistry , Pyruvate Decarboxylase/metabolism , Pyruvic Acid/metabolism , Serum Albumin/metabolism , Substrate SpecificityABSTRACT
The three lipoyl (E2plip) domains of the dihydrolipoyl acetyltransferase component of the pyruvate dehydrogenase (PDH) complex of Escherichia coli house the lipoyl-lysine side chain essential for active-site coupling and substrate channelling within the complex. The structure of the unlipoylated form of the innermost domain (E2plip(apo)) was determined by multidimensional NMR spectroscopy and found to resemble closely that of a nonfunctional hybrid domain determined previously [Green et al. (1995) J. Mol. Biol. 248, 328-343]. The domain comprises two four-stranded beta-sheets, with the target lysine residue residing at the tip of a type-I beta-turn in one of the sheets; the N- and C-termini lie close together at the opposite end of the molecule in the other beta-sheet. Measurement of (15)N NMR relaxation parameters and backbone hydrogen/deuterium (H/D) exchange rates reveals that the residues in and surrounding the lipoyl-lysine beta-turn in the E2plip(apo) form of the domain become less flexible after lipoylation of the lysine residue. This implies that the lipoyl-lysine side chain may not sample the full range of conformational space once thought. Moreover, reductive acetylation of the lipoylated domain (E2plip(holo) --> E2plip(redac)) was accompanied by large changes in chemical shift between the two forms, and multiple resonances were observed for several residues. This implies a change in conformation and the existence of multiple conformations of the domain on reductive acetylation, which may be important in stabilizing this catalytic intermediate.
Subject(s)
Escherichia coli/enzymology , Pyruvate Dehydrogenase Complex/chemistry , Acylation , Lysine/chemistry , Magnetic Resonance Spectroscopy , Models, Molecular , Protein Conformation , Protein Structure, Tertiary , Thermodynamics , Thioctic Acid/chemistryABSTRACT
PURPOSE: This work was carried out to determine the surface tension of block copolymer micelles of 14C labelled ABA poly (oxyethylene-bi-isoprene-b-oxyethylene) which have a long circulating half life in animals. METHODS: The method used was that of phagocytosis. The percentage of micelles phagocytosed by human mononuclear cells was determined in solutions of different surface tension. RESULTS: The values obtained were 72 mN/m which may be predicted for a particle with a long circulating half life in animals. The method also gave an estimate of the surface tension for the mononuclear cells. CONCLUSIONS: This technique has the advantage of determining the surface tension of highly hydrated small particles including stable micelles in an environment similar to that in which they normally exist.
