engleski

Study of subunit interactions in alkaline phosphatase from E. coli using site directed mutagenesis

Although alkaline phosphatase (APase) from E. coli crystallizes as a symmetric dimer, it 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. Rigid parts in the polypeptide structure supposedly accomplish the transfer of conformational information between the active sites across the subunit interface. The importance of the subunit interface and the β -pleated sheet, stretching from underneath an active site to the subunit surface, in the catalytic mechanism has been probed by site-directed mutagenesis. The mutation replacing Thr-81 with alanine and Gln-83 with leucine were introduced into the APase gene. 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. Two kinds of mutant proteins were prepared, a single T81A and double T81A/Q83L mutant. The mutant proteins were purified and compared with wild type APase. Stability of the protein was assessed by thermal denaturation. The affinity for metal ligands and their influence on the quaternary structure was examined by monitoring enzyme inactivation in the presence of EDTA. 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.

site directed mutagenesis; subunit interactions; alkaline phosphatase

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