engleski

Kinetic and dynamic properties of interface mutants of alkaline phosphatase from E.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 a dimeric enzyme with unequal subunits. The proposed model, describing the mechanism of substrate hydrolysis, encompasses a conformational change mediated by subunit interactions. Role and mechanism of possible communication between subunits of this dimmeric enzyme are not resolved. Rigid segments in the polypeptide structure supposedly accomplish 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. Dynamic properties and rigidity of Trp-109 environment of WT APase and mutant enzymes were assessed by acrylamide fluorescence quenching and by measuring phosphorescence lifetime of Trp-109 located in the vicinity of the mutation site and β pleated sheet. Influence of mutations on the kinetic properties determined in 1 M Tris/HCl, pH 8, and in 0.35 M 2A2M1P, pH 10.5 was compared with the effects that mutations had on rigidity of a protein structure.

alkaline phosphatase; dinamyc properties; room temperature phosphorescence

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