Spontaneous Refolding of Escherichia coli Methionine Adenosyltransferase (MetK) Is Extremely Slow, Indicative of Chaperone Dependence.

Escherichia coli methionine adenosyltransferase (MetK) catalyzes the synthesis of S-adenosylmethionine from ATP and l-methionine and is essential for cellular metabolism. To gain insight into its conformational stability and folding behavior in vitro, we investigated the unfolding and refolding of MetK using guanidine hydrochloride (GdnHCl), urea, and acid (pH 2.0) as denaturing conditions. Unfolding was monitored using enzymatic activity, tryptophan fluorescence, far-UV circular dichroism (CD), native-PAGE, and light scattering. GdnHCl, urea, and acid treatment generated structurally distinct denatured ensembles with different structural and functional properties. Although unfolding transitions monitored individually by tryptophan fluorescence and far-UV CD could be described by simple unfolding models, noncoincident transitions together with singular value decomposition (SVD) analysis suggested conformational heterogeneity during unfolding. Refolding of denatured MetK in native buffer was inefficient and accompanied by aggregation, whereas the inclusion of physiologically relevant osmolytes such as glycerol and trehalose substantially improved refolding yields. Complete recovery of enzymatic activity was achieved only from the 8 M urea-denatured state, with a refolding half-time of 4.64 ± 0.23 h. Collectively, these results reveal denaturant-dependent differences in the unfolding and refolding behavior of MetK and highlight the importance of unfolded-state properties and osmolyte-assisted stabilization in determining productive folding outcomes.