engleski

Influence of subunit interface mutations on kinetic and dynamic properties of alkaline phosphatase from E.coli

Mutations, replacing amino acids involved in the formation of hydrogen bonds between subunits of dimeric alkaline phosphatase, have been prepared. Besides establishing its influence on kinetic properties and structural stability, alterations in protein dynamic properties have been studied using room temperature phosphorescence. Kinetic properties of mutant enzymes were virtually the same, differing from the wild type enzyme in the kcat value that was almost twice lower. Changes in rigidity of the protein region of interest was studied by measuring Trp109 phosphoresce lifetime and the rate of its phosphorescence quenching with acrylamide. Changes in protein dynamic properties of mutant proteins compared to the wild type enzyme, as indicated by RTP, did not parallel changes in kinetic properties suggesting that an entropy effect is not responsible for the reduction of kinetic properties. Instead, combined kinetic and dynamic consequences of mutations breaking the hydrogen bonds suggest that breaking of specific links, involved in transmission of conformational change, could be responsible for altered kinetic properties.

alkaline phosphatase ; room temperature phosphorescence ; acrylamide quenching ; kinetic properties ; protein dynamics ; subunit interface

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