engleski

Subunit Cooperation is Required for Activation of Alkaline Phosphatase from E. Coli by Magnesium Ions

Alkaline phosphatase (ALP) is a non-specific phosphomonoesterase catalysing the hydrolysis of a large number of phosphomonoesters. Since ALP is present in almost all forms of life it is assumed that it participates in fundamental biological processes, most probably in phosphate transport. The crystal structure of dimeric enzyme revealed direct participation of two zinc ions in substrate binding and phoshomonoester hydrolysis. Although magnesium strongly activates ALP its role in the kinetic mechanism has remained unexplained. It has been determined that a native ALP contains only 1.3 ą 0.2 moles of Mg2+ per dimer indicating negative cooperativity in Mg2+ binding. In order to establish whether activation by Mg includes cooperative effects we compared activation of active dimer and the enzyme with only one monomer active. Enzyme with only two Zn2+ ions per dimer was prepared and its activation with the Mg2+ was compared with activation of fully active dimer with four Zn2+ ions. Although both enzyme preparation were activated by magnesium much higher concentration of Mg2+ were required to activate monomers indicating that mechanism of activation by magnesium ions includes cooperative effects.

alkaline phosphatase; enzyme kinetics

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