title: Mg2+ binding to alkaline phosphatase correlates with slow changes in protein lability.
abstract: 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.
authors.affiliation: Biophysics Research Division, Department of Biological Chemistry, and Institute of Gerontology, University of Michigan, Ann Arbor, Michigan 48109, USA.
authors.forename: E
authors.initials: E
authors.lastname: Dirnbach
authors.forename: D G
authors.initials: DG
authors.lastname: Steel
authors.forename: A
authors.initials: A
authors.lastname: Gafni
mesh_terms: D000469:Alkaline Phosphatase / Q000187:drug effects / Q000378:metabolism*; D001051:Apoenzymes / Q000378:metabolism; D002413:Cations, Divalent / Q000378:metabolism; D004795:Enzyme Stability; D019791:Guanidine / Q000494:pharmacology; D020035:Holoenzymes / Q000378:metabolism; D008274:Magnesium / Q000378:metabolism*; D008956:Models, Chemical; D008958:Models, Molecular; D011489:Protein Denaturation; D017510:Protein Folding; D011994:Recombinant Proteins / Q000187:drug effects / Q000378:metabolism; D015032:Zinc / Q000378:metabolism
chemical_list: D001051:Apoenzymes; D002413:Cations, Divalent; D020035:Holoenzymes; D011994:Recombinant Proteins; D000469:Alkaline Phosphatase; D008274:Magnesium; D015032:Zinc; D019791:Guanidine