title: Protein mobility inside pyruvate dehydrogenase complexes as reflected by laser-pulse fluorometry. A new approach to multi-enzyme catalysis.
abstract: The fluorescence decay curves of the flavin in all pyruvate dehydrogenase complexes studied here are consistent with a two-exponential fit. One of the lifetimes calculated is very short, as demonstrated by experiments in which a mode-locked argon-ion laser was used for excitation. In three complexes out of the four which were investigated, about equal weights for the amplitudes of the two lifetimes are found. In the three-component complex from Azotobacter vinelandii this is not the case. No effects of the protein concentration on the lifetimes of the fluorophore were found in the concentration range studied. A small but significant difference in lifetime is observed for the A. vinelandii complexes when coenzyme-free complex is compared with complex to which Mg2+ and thiamin diphosphate are added. The correlation time calculated from the polarized decay of the flavin fluorescence at 11 degrees C is around 40 ns and 50 ns for A. vinelandii complexes and Escherichia coli complexes respectively. This correlation time is of the same order as the rotational correlation time of free lipo-amide dehydrogenase itself, but much shorter than would be expected from the molecular weights of the complexes. Models explaining the two lifetimes are discussed. A catalytic mechanism based on the internal mobility of the lipoamide dehydrogenase inside the multi-enzyme complex is proposed.
mesh_terms: D001395:Azotobacter / Q000201:enzymology; D001426:Bacterial Proteins / Q000502:physiology*; D002384:Catalysis; D004926:Escherichia coli / Q000201:enzymology; D005470:Fluorometry / Q000379:methods*; D007834:Lasers; D008274:Magnesium / Q000032:analysis; D008954:Models, Biological; D011768:Pyruvate Dehydrogenase Complex / Q000032:analysis*; D013835:Thiamine Pyrophosphate / Q000032:analysis
chemical_list: D001426:Bacterial Proteins; D011768:Pyruvate Dehydrogenase Complex; D008274:Magnesium; D013835:Thiamine Pyrophosphate