Glutamate dehydrogenase from the thermoacidophilic archaebacterium Sulfolobus solfataricus: studies on thermal and guanidine-dependent inactivation.
The hexameric NAD(P)-dependent glutamate dehydrogenase isolated from the thermoacidophilic archaebacterium Sulfolobus solfataricus shows a remarkable thermal stability which is strictly dependent on protein concentration (half-life at 95 degrees C is 0.25 h and 0.5 h at 0.4 and 0.8 mg/ml, respectively). Temperature-dependent inactivation of the enzyme is apparently irreversible; this process is accompanied by a progressive increase in hydrophobic surface area which leads to protein precipitation. 3 M GdnHCl increases the half-life of the enzyme at 90 degrees C and 0.2 mg/ml 6-fold. The hexamer is the only soluble molecular species revealed by glutaraldehyde fixation after thermal inactivation. Lyotropic salts strongly affect the enzyme thermal stability: the half-life at 90 degrees C and 0.2 mg/ml protein concentration increases more than 6-fold in the presence of 0.4 M Na2SO4 and decreases 4-fold in the presence of 0.4 M NaSCN. The maximum protein thermal stability is observed around the isoelectric pH, between pH 5.2 and pH 6.8. Guanidine-dependent inactivation of the enzyme at 20 degrees C is irreversible above 1.5 M GdnHCl. The decline in percentage of reactivation closely parallels the structural changes detected by fluorescence and the loss of hexameric structure accompanied by the dissociation to monomers, as indicated by glutaraldehyde fixation.
Consalvi
V
V
Dipartimento di Scienze Biochimiche, Università La Sapienza, Roma, Italy.
Chiaraluce
R
R
Politi
L
L
Pasquo
A
A
De Rosa
M
M
Scandurra
R
R
D004789:Enzyme Activation / Q000187:drug effects; D004795:Enzyme Stability; D005453:Fluorescence; D005969:Glutamate Dehydrogenase / Q000378:metabolism*; D005976:Glutaral; D019791:Guanidine; D006146:Guanidines / Q000494:pharmacology*; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D011489:Protein Denaturation; D016944:Sulfolobus / Q000201:enzymology*; D013816:Thermodynamics
D006146:Guanidines; D005969:Glutamate Dehydrogenase; D019791:Guanidine; D005976:Glutaral