TY - JOUR
T1 - Structural studies of a Phe256Trp mutant of human salivary α-amylase
T2 - Implications for the role of a conserved water molecule in enzyme activity
AU - Ramasubbu, Narayanan
AU - Sundar, Krishnan
AU - Ragunath, Chandran
AU - Rafi, Mohamed M.
N1 - Funding Information: We thank Dr. K.I. Varughese, The Scripps Research Institute, for X-ray data collection. This project was supported by the USPHS Grant DE12585 (N.R.).
PY - 2004/1/1
Y1 - 2004/1/1
N2 - 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 2.1Å 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.
AB - 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 2.1Å 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.
KW - Complex
KW - Crystal structure
KW - Mutagenesis and water chain
KW - Salivary α-amylase
KW - Starch hydrolysis
UR - https://www.scopus.com/pages/publications/0346848906
UR - https://www.scopus.com/inward/citedby.url?scp=0346848906&partnerID=8YFLogxK
U2 - 10.1016/j.abb.2003.10.007
DO - 10.1016/j.abb.2003.10.007
M3 - Article
C2 - 14678792
SN - 0003-9861
VL - 421
SP - 115
EP - 124
JO - Archives of Biochemistry and Biophysics
JF - Archives of Biochemistry and Biophysics
IS - 1
ER -