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 introduced. Influence of mutations on kinetic properties and structural stability of mutant enzymes was established. In addition, alterations in protein dynamic properties have been studied using room temperature phosphorescence. Kinetic properties of both mutant enzymes were virtually the same, differing from the wild type enzyme in the kcat value that was almost twice lower. Changes in protein dynamic properties of mutant proteins, compared to the wild type enzyme, did not parallel changes in kinetic properties suggesting that an alteration in the rigidity of the Trp109 environment is not responsible for the reduction of kinetic properties. Instead, combined kinetic and dynamic consequences of introduced mutations suggest that breaking of specific links, involved in transmission of conformational change, could be responsible for altered kinetic properties. (doi: 10.5562/cca2168) Keywords: room temperature phosphorescence, acrylamide quenching, kinetic properties, protein dynamics, subunit interface