engleski

Dimer assymetry and the catalytic cycle of alkaline phosphatase from Escherichia coli

Although alkaline phosphatase (APase) from E. coli crystallizes as a symmetric dimer, it displays deviations from Michaelis-Menten kinetics, supported by a model describing dimeric enzyme with unequal subunits [Orhanović S., Pavela-Vrančič M., and Flogel-Mršić M., (1994) Acta. Pharm. 44, 87-95]. The possibility, that the observed asymmetry could be attributed to negative cooperativity in Mg(II) binding, has been examined. The influence of the metal ion content on the catalytic properties of APase from E. coli has been examined by kinetic analysis. An activation study has indicated that Mg(II) enhances APase activity by a mechanism that involves interactions between subunits. The observed deviations from Michaelis-Menten kinetics are independent of saturation with Zn(II) or Mg(II) ions, suggesting that asymmetry is an intrinsic property of the dimeric enzyme. In accordance with the experimental data, a model describing the mechanism of substrate hydrolysis by APase has been proposed. The release of the product is enhanced by a conformational change generating a subunit with lower affinity for both the substrate and the product. In the course of the catalytic cycle the conformation of the subunits alternates between two states in order to enable substrate binding, and product release. APase displays higher activity in the presence of Mg(II), since binding of Mg(II) increases the rate of conformational change. A conformationally controlled and Mg(II)-assisted dissociation of the reaction product (Pi) could serve as a kinetic switch preventing loss of Pi into the environment.

metalloenzymes; conformational change; subunit interactions; enzyme asymmetry; phosphate metabolism

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