Urea-induced inactivation and denaturation of clostridial glutamate dehydrogenase: the absence of stable dimeric or trimeric intermediates.
Urea-induced effects in clostridial glutamate dehydrogenase (GDH, EC 1.4.1.2) were studied by spectrophotometry, circular dichroism, FPLC, affinity chromatography and PAGE. Denaturation of enzyme occurred over a narrow range of urea concentrations (2.5-3.5 M), accompanied by inactivation of enzyme with a similar rate constant. The contribution of instantaneous inhibition by urea was also ascertained. FPLC studies of urea-treated GDH gave no evidence for dissociated oligomeric fragments of the hexamer in the presence of subdenaturing concentrations of urea. Likewise a mixture of fully 5,5'-dithiobis-(2-nitrobenzoic acid)-modified GDH hexamers and unmodified enzyme in 2 M urea failed to give rise to hybrid molecules. Exposure of unmodified GDH to high concentrations of urea led to the dissociation of hexamers to denatured monomers followed by association to form non-specific high-M(r) aggregates. This conclusion was confirmed by native gradient PAGE experiments. Various specific ligands stabilized the enzyme against urea-induced inactivation, succinate and 2-oxoglutarate being particularly effective. This protection of the native state was enhanced in ternary complexes, and the complex most resistant to urea-induced inactivation was the productive ternary complex GDH-NADH-2-oxoglutarate. Native gradient PAGE experiments indicate that these protecting ligands preserve the native hexameric structure of GDH.
D002853:Chromatography, Liquid; D002942:Circular Dichroism; D003013:Clostridium / Q000201:enzymology*; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation / Q000187:drug effects; D005969:Glutamate Dehydrogenase / Q000737:chemistry / Q000187:drug effects* / Q000378:metabolism*; D007700:Kinetics; D046911:Macromolecular Substances; D011487:Protein Conformation; D011489:Protein Denaturation / Q000187:drug effects; D012680:Sensitivity and Specificity; D013329:Structure-Activity Relationship; D014508:Urea / Q000494:pharmacology*
D046911:Macromolecular Substances; D014508:Urea; D005969:Glutamate Dehydrogenase