Role of magnesium in Escherichia coli alkaline phosphatase.
Alkaline phosphatase of E. coli, isolated by procedures which do not alter its intrinsic metal content, contains 1.3 +/- 0.3 g-atom of magnesium and 4.0 +/- 0.2 g-atom of zinc per molecule of molecular weight 89,000. Magnesium, the role of which has been unappreciated, significantly affects the function and structure of alkaline phosphatase containing either 2 or 4 g-atom of zinc per mole. Magnesium does not activate the apoenzyme but increases the activity of the enzyme containing 2 g-atom of zinc 4.4-fold and that of the enzyme containing 4 g-atom 1.2-fold. The results obtained with enzyme in which cobalt is substituted for zinc are analogous. Moreover, the absorption and electron paramagnetic resonance spectra of cobalt phosphatases reveal the effects of magnesium on cobalt coordination geometry. Addition of magnesium changes the spectral characteristics of the apoenzyme reconstituted with 2 g-atom of cobalt from predominantly octahedral to 4- or 5-coordinate geometry. These two classes of cobalt binding sites have been associated with catalysis and structure stabilization, respectively. Therefore, magnesium controls the occupancy of the catalytic and structural binding sites and modulates the resultant enzymatic activity. Hydrogen-tritium exchange was employed to determine the effects of magnesium on the conformational stability of phosphatase. Magnesium stabilizes the dynamic structural properties, both of apophosphatase and of enzyme containing 2 g-atom of zinc, which is further stabilized by 2 more zinc atoms. The role of magnesium and other metal ions in regulatory processes, only now beginning to be explored fully, will likely emerge as an important avenue for achievement of regulatory effects in metalloenzymes.
D000469:Alkaline Phosphatase / Q000032:analysis / Q000378:metabolism*; D001665:Binding Sites; D003035:Cobalt / Q000494:pharmacology; D004926:Escherichia coli / Q000201:enzymology*; D008274:Magnesium / Q000032:analysis / Q000494:pharmacology*; D008970:Molecular Weight; D011485:Protein Binding; D011487:Protein Conformation; D015032:Zinc / Q000032:analysis / Q000494:pharmacology
D003035:Cobalt; D000469:Alkaline Phosphatase; D008274:Magnesium; D015032:Zinc