title: Slow conformational changes of a Neurospora glutamate dehydrogenase studied by protein fluorescence.
abstract: 1. The NADP-dependent glutamate dehydrogenase of Neurospora crassa undergoes slow reversible structural transitions, with half-times in the order of a few minutes, between active and inactive states. The inactive state of the enzyme, which predominates at pH values below 7.0, has an intrinsic tryptophan fluorescence 25% lower than that of the active state, which predominates at pH values above 7.6. The inactive state can be activated either by an increase in pH or by addition of activators such as succinate. 2. The kinetics of the slow transitions that follow activating and inactivating rapid changes in conditions have been monitored by measurements of protein fluorescence. The results show that the slow reversible conformational change detected by the change in fluorescence is the rate-limiting process for enzyme activation and inactivation. 3. In both directions this conformational change follows apparent first-order kinetics and the rate constant is independent of protein concentration. These kinetics and published measurements of molecular weight are indicative of an isomerization process. 4. In both directions the changes show a large energy of activation and a large positive entropy of activation, consistent with a considerable disturbance of conformation in the transition state. 5. Comparisons of the fluorescence emission spectra of the active and inactive states indicate that the difference in fluorescence is produced by quenching, possibly intramolecular, in the inactive conformation. Iodide ions cause similar quenching. 6. In some mutationally altered forms of the enzyme comparable but modified conformational changes can be followed by protein fluorescence.
mesh_terms: D004789:Enzyme Activation; D005453:Fluorescence; D005969:Glutamate Dehydrogenase; D006863:Hydrogen-Ion Concentration; D007536:Isomerism; D007700:Kinetics; D008970:Molecular Weight; D009249:NADP; D009491:Neurospora / Q000201:enzymology*; D009492:Neurospora crassa / Q000201:enzymology*; D011487:Protein Conformation; D011506:Proteins / Q000032:analysis; D013386:Succinates; D013816:Thermodynamics
chemical_list: D011506:Proteins; D013386:Succinates; D009249:NADP; D005969:Glutamate Dehydrogenase