Allosteric modulation of pyrophosphatase activity of rat osseous plate alkaline phosphatase by magnesium ions.
Pyrophosphatase activity of rat osseous plate alkaline phosphatase was studied at different concentrations of calcium and magnesium ions, with the aim of characterizing the modulation of enzyme activity by these metals. In the absence of metal ions, the enzyme hydrolysed pyrophosphate following "Michaelian" kinetics with a specific activity of 36.7 U/mg and K0.5 = 88 microM. In the presence of low concentrations (0.1 mM) of magnesium (or calcium) ions, the enzyme also exhibited "Michaelian" kinetics for the hydrolysis of pyrophosphate, but a significant increase in specific activity (123 U/mg) was observed, K(m) values remained almost unchanged. Quite different behavior occurred in the presence of 2 mM magnesium (or calcium) ions. In addition to low-affinity sites (K0.5-40 and 90 microM, for magnesium and calcium, respectively), high-affinity sites were also observed with K0.5 values 100-fold lower. The high-affinity sites observed in the presence of calcium ions represented about 10% of those observed for magnesium ions. This was correlated with the fact that only magnesium ions triggered conformational changes yielding a fully active enzyme. These results suggested that the enzyme could hydrolyse pyrophosphate, even at physiological concentrations (4 microM), since magnesium concentrations are high enough to trigger conformational changes increasing the enzyme activity. A model, suggesting the involvement of magnesium ions in the hydrolysis of pyrophosphate by rat osseous plate alkaline phosphatase is proposed.
Leone
F A
FA
Departamento de Química, Faculdade de Filosofia, Ciências e Letras-USP, Ribeirão Preto, Brazil.
Rezende
L A
LA
Ciancaglini
P
P
Pizauro
J M
JM
D000469:Alkaline Phosphatase / Q000032:analysis / Q000378:metabolism*; D000494:Allosteric Regulation; D000818:Animals; D016025:Bone Transplantation / Q000502:physiology; D001842:Bone and Bones / Q000201:enzymology*; D002118:Calcium / Q000494:pharmacology / Q000502:physiology*; D006868:Hydrolysis; D066298:In Vitro Techniques; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology / Q000502:physiology*; D010710:Phosphates / Q000737:chemistry; D011755:Pyrophosphatases / Q000378:metabolism*; D051381:Rats; D013050:Spectrometry, Fluorescence
D010710:Phosphates; D000469:Alkaline Phosphatase; D011755:Pyrophosphatases; D008274:Magnesium; D002118:Calcium