The effect of Mg(II) on the spectral properties of Co(II) alkaline phosphatase.
Alkaline phosphatase of Escherichia coli, isolated by procedures which do not alter its intrinsic metal content, contains 1.3 +/- 0.3 g-atom(s) of magnesium and 4.0 +/- 0.2 g-atoms of zinc per mol of molecular weight 89 000 (Bosron et al., 1975). Substitution of Co(II) for Zn(II) and/or Mg(II) results in spectral properties which can be correlated with enzymatic activity. Magnesium does not activate the apoenzyme but augments the activity of 2-Co(II) enzyme almost 3-fold and that of the 4-Co(II) enzyme 1.3-fold. The magnesium-induced increase in activity of the 2-Co(II) enzyme is accompanied by spectral changes which are consistent with an alteration from largely octahedral-like to pentacoordinate-like coordination geometry. Magnesium increases the intensity of the absorption and magnetic circular dichroism (MCD) signals of the 4-Co(II) enzyme but without evidence of changes in coordination geometry. Cobalt when bound to the magnesium sites results in octahedral-like EPR spectra, unperturbed by phosphate which significantly affects cobalt at the pentacoordinate-like sites. In the absence of magnesium, 6 g-atoms of cobalt are required to maximize the spectral properties, but activity does not increase further after the addition of only 4 g-atoms of cobalt, while activity is optimal with only 2 g-atoms of cobalt. Hydrogen-tritium exchange measurements indicate that magnesium also stabilizes the dynamic structural properties of the apo- and 2-Co(II) enzymes but has little effect on the structure of 4-Co(II) phosphatase. The response to magnesium of both the spectral properties and enzymatic activities of cobalt alkaline phosphatase demonstrates that magnesium regulates cobalt (and zinc) binding and modulates the activity of the resultant products.
D000469:Alkaline Phosphatase / Q000378:metabolism*; D001051:Apoenzymes / Q000378:metabolism; D001665:Binding Sites; D003035:Cobalt / Q000494:pharmacology*; D004926:Escherichia coli / Q000201:enzymology; D008274:Magnesium / Q000494:pharmacology*; D010710:Phosphates / Q000494:pharmacology; D011485:Protein Binding; D013057:Spectrum Analysis; D014316:Tritium; D015032:Zinc / Q000494:pharmacology
D001051:Apoenzymes; D010710:Phosphates; D014316:Tritium; D003035:Cobalt; D000469:Alkaline Phosphatase; D008274:Magnesium; D015032:Zinc