engleski

Influence of mutations at the subunit interface on kinetic and dynamic properties of alkaline phosphatise from E. coli

Alkaline phosphatase (APase, E.C. 3.1.3.1) is a non-specific phosphomonoesterase catalysing the hydrolysis of a large number of phosphomonoesters. Most unresolved questions, relating to the catalytic mechanism of APase concern the influence of conformational changes and allosteric interactions on catalytic efficiency. APase displays deviations from Michaelis-Menten kinetics, supported by a model describing a dimeric enzyme with unequal subunits. The proposed model, describing the mechanism of substrate hydrolysis, encompasses a conformational change mediated by subunit interactions. The importance of the subunit interface and the β -pleated sheet stretching from underneath an active site to the subunit surface in the transfer of conformational information, has been assessed by site-directed mutagenesis followed by kinetic analysis and probing of dynamic properties of the APase dimer using room temperature phosphorescence. The mutant APase, carrying alanine in place of Thr81, as well as a double mutant carrying alanine in place of Thr81 and leucine in place of Gln83 were analyzed in comparison to the wild type protein. Amino acid residues Thr-81 and Gln-83 are located within the β -pleated sheet at the contact surface between the subunits, and form hydrogen bonds with analogous amino acids from the adjacent subunit. The mutant proteins were purified and compared with wild type APase. Kinetic properties have been determined in 1 M Tris/HCl, pH 8, and in 0.35 M 2A2M1P, pH 10.5. Mutations introduced at the subunit interface affected both structural stability of the protein and its kinetic properties emphasising the importance of subunit interaction in the catalytic mechanism.

alkaline phosphatase; site directed mutagenesis; subunit interface; kinetic properties

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