Extensive site-directed mutagenesis reveals interconnected functional units in the alkaline phosphatase active site

摘要

Enzymes are biological catalysts that speed up the reactions that are essential for life. As such, enzymes convert ‘reactant’ molecules into other molecules. Reactant molecules bind to part of the enzyme called the active site. Some of the amino acids that make up the active site must directly interact with these molecules to catalyze the reaction. Mutating individual active site amino acids often greatly reduces or destroys the ability of the enzyme to increase reaction rates. These amino acids are known as ‘catalytic residues’. However, catalytic residues do not work in isolation instead, they interact with other residues in the enzyme to carry out their function. Therefore, the effects of these interactions need to be characterized in order to fully understand how enzymes work. Sunden et al. explored the interactions within a network of five residues found at the active site of an enzyme, called alkaline phosphatase, which was taken from the bacterial species E. coli. Nearly all of the possible combinations of these five residues were examined. The results of these experiments indicated that even though all five residues are structurally linked, only a subset of the residues affected one another functionally, even though all of them are structurally connected. In particular, three groups—or functional units—of residues were found in the enzyme structure. The residues within each functional unit directly or indirectly cooperate to increase different aspects of the enzyme's catalytic activity. Sunden et al. used this information to develop models that describe how the functional units work together, and suggest that the likelihood of the active site evolving so that its residues are not fully cooperative is high. It remains to be seen whether similar cooperative networks exist in the active sites of other enzymes and how residues further away affect those in and around the active site. Understanding how the residues in the active site work together and being able to model their interactions could help efforts to develop more efficient enzymes for use in biotechnology in the future.