The mechanism of hydrolysis of beta-glycerophosphate by kidney alkaline phosphatase.
1. To identify the functional groups that are involved in the conversion of beta-glycerophosphate by alkaline phosphatase (EC 3.1.3.1) from pig kidney, the kinetics of alkaline phosphatase were investigated in the pH range 6.6-10.3 at substrate concentrations of 3 muM-30 mM. From the plots of log VH+ against pH and log VH+/KH+m against pH one functional group with pK = 7.0 and two functional groups with pK = 9.1 were identified. These groups are involved in substrate binding. Another group with pK = 8.8 was found, which in its unprotonated form catalyses substrate conversion. 2. GSH inhibits the alkaline phosphatase reversibly and non-competitively by attacking the bound Zn(II). 3. The influence of the H+ concentration on the activation by Mg2+ ions of alkaline pig kidney phosphate was investigated between pH 8.4 and 10.0. The binding of substrate and activating Mg2+ ions occurs independently at all pH values between 8.4 and 10.0. The activation mechanism is not affected by the H+ concentration. The Mg2+ ions are bound by a functional group with a pK of 10.15. 4. A scheme is proposed for the reaction between enzyme, substrate, Mg2+ and H+ and the overall rate equation is derived. 5. The mechanism of substrate binding and splitting by the functional groups of the active centre is discussed on the basis of a model. Mg2+ seems to play a role as an autosteric effector.
D000469:Alkaline Phosphatase / Q000302:isolation & purification / Q000378:metabolism*; D000818:Animals; D001665:Binding Sites; D005978:Glutathione / Q000378:metabolism; D005994:Glycerophosphates / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007668:Kidney / Q000201:enzymology; D007700:Kinetics; D008274:Magnesium; D008956:Models, Chemical; D013552:Swine; D015032:Zinc / Q000378:metabolism
D005994:Glycerophosphates; D000469:Alkaline Phosphatase; D005978:Glutathione; D008274:Magnesium; D015032:Zinc