Investigation of the quaternary structure of Neurospora pyruvate kinase by cross-linking with bifunctional reagents: the effect of substrates and allosteric ligands.
Pyruvate kinase (EC 2.7.1.40) of Neurospora, a tetramer composed of apparently identical subunits, has been shown to be a dimer of dimers by interprotomeric cross-linking experiments in which bifunctional reagents were used. An analysis of the polyacrylamide gel profiles of the enzyme after cross-linking with glutaraldehyde, dimethyl suberimidate, and dimethyl adipimidate shows that the extent of intersubunit cross-linking is influenced markedly by the ligand bound to the enzyme. Bifunctional cross-linking reagents with a shorter distance between the two functional groups form cross-links effectively in the unliganded enzyme. In the FDP-pyruvate kinase complex, cross-linking was observed over longer distances compared with the unliganded enzyme. It is demonstrated that covalent cross-linkers cah be used as sensitive indicators of conformational changes induced in pyruvate kinase by substrates and allosteric ligands.
D000272:Adipates; D000494:Allosteric Regulation; D000495:Allosteric Site; D004591:Electrophoresis, Polyacrylamide Gel; D005976:Glutaral; D007094:Imides; D008024:Ligands; D046911:Macromolecular Substances; D008970:Molecular Weight; D009491:Neurospora / Q000201:enzymology*; D009492:Neurospora crassa / Q000201:enzymology*; D011485:Protein Binding; D011487:Protein Conformation; D011770:Pyruvate Kinase / Q000378:metabolism
D000272:Adipates; D007094:Imides; D008024:Ligands; D046911:Macromolecular Substances; D011770:Pyruvate Kinase; D005976:Glutaral