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.

Glutamate dehydrogenase from the thermoacidophilic archaebacterium Sulfolobus solfataricus

“…Under our assay conditions, it is likely that the diluted enzyme undergoes slow conformational changes induced by ligands and physical agents as already reported for other glutamate dehydrogenases [43,45,461. This would produce the kinetic appearance of a mixture of enzymes catalysing the same reaction [49].…”

“…As in the case of the ox enzyme [43], abrupt transition in the double-reciprocal plot may be generated by more complex reciprocal initial-rate equation than Eqn (1). This type of data might be given by a multi-site enzyme which exists in two or more conformational states that equilibrate only very slowly under the effects of various physical agents like temperature or pH in the presence of ligands [13,43,45,461.…”

Crystallization and preliminary X-ray analysis of the NADP-specific glutamate dehydrogenase fromNeurospora crassa

“…The NADP-specific enzyme of N. crassa is of interest as it is allosterically regulated at the physiological pH of 7.2 by the substrate 2-oxoglutarate and other TCA (tricarboxylic acid) cycle intermediates (West, Tuveson, Barrat & Fincham, 1967;Ashby, Wootton & Fincham, 1974). Mutants of N. crassa defective in amination, and therefore termed am mutants, were first characterized from the Beadle and Tatum collection of auxotrophs (Fincham, 1950(Fincham, , 1951.…”

Oxidation of Neurospora crassa NADP-specific glutamate dehydrogenase by activated oxygen species

“…The experiments with substrates and products were done at pH 7, at which GDH(NADP) is in an inactive form in the absence of substrates or in the presence of NADPH (52). At this pH, the enzyme shifts to an active conformation in the presence of 2-oxoglutarate or 2-oxoglutarate plus NADPH (2,52). No clear-cut conclusion could be drawn from these results to indicate that either the active or the inactive conformation of the enzyme was more liable to inactivation by activated oxygen species.…”

“…The active form is found in the presence of 2-oxoglutarate, NADP or 2-oxoglutarate plus NADPH or NADP, or in the presence of glutamate (52). Binding of NADPH shifts the equilibrium to the inactive conformation of the enzyme (2). The effect of the substrates and products upon the oxidation of GDH(NADP) by ascorbate, Fe3+, and EDTA is shown in Fig.…”