The in vitro reactivation of unfolded Escherichia coli alkaline phosphatase (AP) in the presence of the two natively bound metals Zn2+ and Mg2+ produces two protein species, characterized by different guanidine hydrochloride denaturation kinetics. The high-lability AP form slowly converts to the low-lability form in a first-order reaction with a characteristic lifetime (inverse rate constant) of approximately 300 h at pH 8.0 and 25 degrees C. Addition of Zn2+ and Mg2+ ligands to (folded) apo-AP also produces two protein species, with denaturation kinetics and a long conversion lifetime similar to those found in refolding AP. In contrast, adding Zn2+ alone to apo-AP produces only the high-lability species with no subsequent structural change, suggesting that Mg2+ binding is the event which is responsible for the production of the low-lability AP. The rate of conversion from high- to low-lability AP was found to be linearly dependent on Mg2+ concentration, indicating that Mg2+ binding is rate limiting for this reaction. Experiments where either Zn2+ or Mg2+ was added first, with the second metal added later, show that Mg2+ binding is slowed by the prior presence of bound Zn2+. Mg2+ binding to Zn-AP also slightly increases the enzymatic activity; however, the extent of formation of the low-lability species is related to the square of the Mg2+-induced activity increase. Thus the binding of two Mg2+ to AP produces the dramatic reduction in the rate of denaturation that characterizes the low-lability species. The data suggest the possibility of long distance intersubunit interactions and a role for Mg2+ in providing "kinetic stability" for AP.

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The in vitro folding of Escherichia coli alkaline phosphatase (AP) from the guanidine hydrochloride (GdnHCl) denatured state is characterized by a significant slow phase in the post activational recovery of native protein lability (probed by the susceptibility to GdnHCl denaturation and occurring on the time scale of days) as well as a slow phase in the recovery of activity (on the time scale of minutes). Slow folding events have often been attributed to cis-trans isomerizations of XPro peptide bonds, a plausible explanation for AP, which contains 21 prolines per subunit. To investigate the role of proline isomerization in the two measures of refolding mentioned above, we have performed "doublejump" GdnHCl denaturation/renaturation experiments, with a third jump, where the rate of unfolding of refolded protein upon exposure to denaturant was added to assess the rate of change of lability. Our measurements of the time evolution of both the lability and the reactivation of refolded AP as a function of denaturation time show that proline isomerization is unlikely to be the cause of either of these slow events in the refolding of AP. The conclusions are further confirmed by the absence of proline isomerization effects when AP is refolded in the presence of human and periplasmic E. coli peptidyl-prolyl isomerase.Numerous studies of protein folding have shown that kinetic trapping can produce folded forms of the protein that differ from the native state and may in fact lead to long time scale changes in structure. The molecular origin of this behavior and the nature of the trapped state are unclear and of considerable interest in studies of folding. For example, in vitro refolded ␤-lactoglobulin adopts a non-native conformation, as shown both by antibody binding (1) and by room temperature phosphorescence and lability (susceptibility to GdnHCl 1 denaturation) studies (2). ␣-Lytic protease and subtilisin both require propeptides as intramolecular chaperones for successful folding, and the folding of either protein without the propeptide results in a stable molten globule structure that can convert to the native conformation only with the addition of the propeptide region in trans (3, 4). If subtilisin is folded with a mutated propeptide, it adopts a distinctly altered conformation (4). In these cases, the protein structure appears to be confined indefinitely in a near-native state. However, in other cases slow structural changes toward the native state have been observed. For example, in vivo newly synthesized phosphoglycerate kinase adopts a conformation that is slowly converted to a more stable state over time (5, 6). When the "old" (stable) form is unfolded in vitro, it adopts the "young" conformation upon initial refolding and then spontaneously recovers the old state (7, 8). The difference between stable, non-native state, kinetic trapping and slow conformational shifting toward the native state is likely due to the height of the activation energy barrier directly between the states, with a high barrier rendering ...

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