Involvement of single residue tryptophan 548 in the quaternary structural stability of pigeon cytosolic malic enzyme

Pigeon cytosolic malic enzyme has a double dimer quaternary structure with three tryptophanyl residues in each monomer distributed in different structural domains. The enzyme showed a three-state unfolding phenomenon upon increasing the urea concentration (Chang, H. C., Chou, W. Y., and Chang, G. G. (2002) J. Biol. Chem. 277, 4663-4671). At urea concentration of 4-4.5 M, where the intermediate form was detected, the enzyme existed as partially unfolded dimers, which were easily polymerized. Mn2+ provided full protection against the polymerization. To further characterize this phenomenon, three mutants of the enzyme (W129, W321, and W548), each with only one tryptophanyl residue left, were constructed. All these mutants were successfully overexpressed in Escherichia coli cells and purified to homogeneity. Changes in the circular dichroism spectra of all mutants revealed a three-state urea-unfolding process in the absence of Mn2+. In the presence of 4 mM Mn2+, W548 and wild type (WT) enzymes shifted to monophasic, while W129 and W321 were still biphasic. Similar results were obtained from the fluorescence spectral changes, except for W321, which showed monophasic denaturation curve with or without Mn2+. Analytical ultracentrifugation analysis indicated that the mutant enzymes were polymerized at 4.5 M urea, and Mn2+ provided protective effect on W548 and WT enzymes only. Other mutants with mutated Trp-548 polymerized at 4.5 M urea in the absence or presence of 4 mM Mn2+. The above results indicate that a single residue, Trp-548, in the subunit interface region, is responsible for the integrity of the quaternary structure of the pigeon cytosolic malic enzyme.