Limited proteolysis and proton NMR spectroscopy of Bacillus stearothermophilus pyruvate dehydrogenase multienzyme complex.
The pyruvate dehydrogenase multienzyme complex of Bacillus stearothermophilus was treated with chymotrypsin at pH 7 and 0 degrees C. Loss of the overall catalytic activity lagged behind the rapid cleavage of the lipoate acetyltransferase polypeptide chains, whose apparent Mr fell from 57 000 to 45 000 as judged by sodium dodecylsulphate/polyacrylamide gel electrophoresis. The inactive chymotrypsin-treated enzyme had lost the lipoic-acid-containing regions of the lipoate acetyltransferase chains, yet remained a highly assembled structure. Treatment of this chymotryptic core complex with trypsin at pH 7.0 and 0 degrees C caused a further shortening of the lipoate acetyltransferase polypeptide chains to an apparent Mr of 28 000 and was accompanied by disassembly of the complex. The lipoic-acid-containing regions are therefore likely to be physically exposed in the intact complex, protruding from the structural core formed by the lipoate acetyltransferase component between the subunits of the other component enzymes. Proton nuclear magnetic resonance spectroscopy demonstrated that the enzyme complex contains large regions of polypeptide chain with remarkable intramolecular mobility, most of which were retained after excision of the lipoic-acid-containing regions with chymotrypsin. It is likely that the highly mobile regions are in the lipoate acetyltransferase component and facilitate movement of the lipoic acid residues. Such polypeptide chain mobility provides the molecular basis of a novel system of active-site coupling in the 2-oxo acid dehydrogenase multienzyme complexes.
D001426:Bacterial Proteins / Q000302:isolation & purification*; D001411:Geobacillus stearothermophilus / Q000201:enzymology*; D006868:Hydrolysis; D009682:Magnetic Resonance Spectroscopy; D008854:Microscopy, Electron; D011522:Protons; D011768:Pyruvate Dehydrogenase Complex / Q000302:isolation & purification*
D001426:Bacterial Proteins; D011522:Protons; D011768:Pyruvate Dehydrogenase Complex