Catalytic properties of alkaline phosphatase from pig kidney.
The enzymic properties of alkaline phosphatase (EC 3.1.3.1) from pig kidney brush-border membranes were studied. 1. It hydrolyses ortho- and pyro-phosphate esters, the rate limiting step (V(max.)) being independent of the substrate. It transphosphorylates to Tris at concentrations above 0.1m-Tris. 2. The pH optimum for hydrolysis was between 9.8 and 10. The pK of the enzyme-substrate complex is 8.7 for p-nitrophenyl phosphate and beta-glycerophosphate. Excess of substrate inhibits the enzymic activity with decreasing pH. The pK of the substrate-inhibited enzyme-substrate complex, 8.7, is very similar to that for the enzyme-substrate complex. The pK values of the free enzyme appear to be 8.7 and 7.9. 3. Inactivation studies suggest that there is an essential tyrosine residue at the active centre of the enzyme. 4. The energy of activation (E) and the heat of activation (DeltaH) at pH9.5 showed a transition at 24.8 degrees C that was unaffected by Mg(2+). 5. Kinetic and atomic-absorption analysis indicated the essential role of two Zn(2+) ions/tetrameric enzyme for an ordered association of the monomers. Zn(2+) in excess and other bivalent ions compete for a second site with Mg(2+). Mg(2+) enhances only the rate-limiting step of substrate hydrolysis. 6. Amino acid inhibition studies classified the pig kidney enzyme as an intermediate type of previously described alkaline phosphatases. It has more similarity with the enzyme from liver and bone than with that from placenta.
D000469:Alkaline Phosphatase / Q000037:antagonists & inhibitors; D000596:Amino Acids; D000818:Animals; D011756:Diphosphates / Q000378:metabolism; D004789:Enzyme Activation; D005994:Glycerophosphates; D006863:Hydrogen-Ion Concentration; D007668:Kidney / Q000201:enzymology*; D007700:Kinetics; D008274:Magnesium; D008566:Membranes / Q000201:enzymology; D009596:Nitrophenols; D010710:Phosphates / Q000378:metabolism; D010770:Phosphotransferases; D013054:Spectrophotometry, Atomic; D013552:Swine; D013696:Temperature; D015032:Zinc
D000596:Amino Acids; D011756:Diphosphates; D005994:Glycerophosphates; D009596:Nitrophenols; D010710:Phosphates; D010770:Phosphotransferases; D000469:Alkaline Phosphatase; D008274:Magnesium; D015032:Zinc