Mg2+ binding to alkaline phosphatase correlates with slow changes in protein lability.
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.
Dirnbach
E
E
Biophysics Research Division, Department of Biological Chemistry, and Institute of Gerontology, University of Michigan, Ann Arbor, Michigan 48109, USA.
Steel
D G
DG
Gafni
A
A
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
D001051:Apoenzymes; D002413:Cations, Divalent; D020035:Holoenzymes; D011994:Recombinant Proteins; D000469:Alkaline Phosphatase; D008274:Magnesium; D015032:Zinc; D019791:Guanidine