Domain structure of isocitrate dehydrogenase from Azotobacter vinelandii.

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1 Isocitrate dehydrogenase from Azotobacter Vinelandii, which consists of a single polypeptide chain of Mr 80,000, is completely unfolded in 3 M guanidine hydrochloride at pH 7.5 and 22 C as judge by hydrodynamic criteria and a study of tryptophan fluorescence. 2 Transfer of denatured isocitrate dehydrogease to native solution conditions (less than 0.1 M guanidine hydrochloride) leads to rapid reactivation (t1/2 ∼ 1 min at 22 C,pH 7.5). Practically complete reactivation (85-95%) can be achieved under properly chosen conditions. 3 At high protein concentration(c > 0.5 - 1.0 mg/ml) intermolecular aggregation competes with proper folding and reactivation. Renatured isocitrate dehydrogenase is indistinguishable from the original native enzyme provided that aggregated material is removed by centrifugation and gel filtration. 4 The denaturation by guanidine hydrochloride proceeds in two separable steps: N⇄D1⇄D2.The midpoint of the N⇄ D1 transition is at 0.3 M, that of the D1⇄ D2 transition at 1.2-1.5 M guanidine hydrochloride. The sedimentation coefficient (s20,w) drops from 4.5 S for the native conformation N to 4.5 S for D1 and 2.3 S for D2 The relative values for the tryptophan fluorescence of D1 and D2 are 50% and 25% of that of N, respectively (γex = 295nm, γem = 330nm). 5 Protease digestion studies using thermolysin or trypsin or trypsin in 0.4 M guanidine hydrochloride suggest that isocitrate dehydrogenase from A vinelandii is composed of three domains which, according to their molecular masses, are designated as the 45-kDa and the 19-kDa, the 16-kDa domains. The 45-kDa and 16-kDa domains are resistant to protease digestion in 0.4 M guanidine hydrochloride, while the 19-kDa domain is rapidly digested under these conditions. 6 The(un)folding intermediate D1 thus represents a conformation comprising folded 45-kDa and 16-kDa domains and a largely unfolded 19-kDa domain. In D2 all three domains have unfolded. The observation that intermolecular aggregation is favored by low concentrations of guanidine hydrochloride (0.3-0.5 M) indicates that this process may depend on intermolecular interactions between the 45-kDa and 16-kDa domains.