Structural studies of a Phe256Trp mutant of human salivary α-amylase: implications for the role of a conserved water molecule in enzyme activity

In the mechanism of hydrolysis of starch by α-amylases, a conserved water molecule bridging two catalytic residues has been implicated. In human salivary α-amylase (HSAmy), this water (W641), observed in many α-amylase structures, is part of a chain of water molecules. To test the hypothesis that W641 may be involved in the mechanism, Phe256 in the close vicinity was mutated to a Trp residue. X-ray structure of F256W complexed to 2-amino-2-(hydroxyethyl)-1,3-propanediol at 2.1 Å revealed that the water chain is disrupted. In the F256W structure exhibits a positional shift in His305, characteristic of α-amylase complex structures. Kinetic analysis, in comparison with HSAmy, revealed that the mutant exhibited a 70-fold decrease in the specific activity for starch and significantly reduced kcat (20-fold) and Km (4-fold) for maltoheptaoside. Collectively, these results suggest that W641 and the chain of water molecules may be critical for the α-amylase activity.