Abstract
The globular 22-kDa protein UMP/CMP from Dictyostelium discoideum (UmpK) belongs to the family of nucleoside monophosphate (NMP) kinases. These enzymes not only show high sequence and structure similarities but also share the α/β-fold, a very common protein topology. We investigated the protein folding mechanism of UmpK as a representative for this ubiquitous enzyme class. Equilibrium stability towards urea and the unfolding and refolding kinetics were studied by means of fluorescence and far-UV CD spectroscopy. Although the unfolding can be described by a two-state process, folding kinetics are rather complex with four refolding phases that can be resolved and an additional burst phase. Moreover, two of these phases exhibit a pronounced rollover in the refolding limb that cannot be explained by aggregation. Whilst secondary structure formation is not observed in the burst phase reaction, folding to the native structure is strongly influenced by the slowest phase, since 30% of the α-helical CD signal is restored therein. This process can be assigned to proline isomerization and is strongly accelerated by the Escherichia coli peptidyl-prolyl isomerase trigger factor. The analysis of our single-mixing and double-mixing experiments suggests the occurrence of an off-pathway intermediate and an unproductive collapsed structure, which appear to be rate limiting for the folding of UmpK.
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