1Q4N

Structural studies of Phe256Trp of human salivary alpha-amylase: implications for the role of a conserved water molecule and its associated chain in enzyme activity

Summary for 1Q4N

• Entry DOI: 10.2210/pdb1q4n/pdb
• Descriptor: Alpha-amylase, salivary, CALCIUM ION, CHLORIDE ION, ... (5 entities in total)
• Functional Keywords: amylase, mutagenesis, tris, inhibitor, hydrolase
• Biological source: Homo sapiens (human)
• Total number of polymer chains: 1
• Total formula weight: 56233.04

Authors

Ramasubbu, N. (deposition date: 2003-08-04, release date: 2004-03-16, Last modification date: 2024-11-06)

Primary citation

Ramasubbu, N.,Sundar, K.,Ragunath, C.,Rafi, M.M.
Structural studies of a Phe256Trp mutant of human salivary alpha-amylase: implications for the role of a conserved water molecule in enzyme activity
Arch.Biochem.Biophys., 421:115-124, 2004

PubMed Abstract: In the mechanism of hydrolysis of starch by alpha-amylases, a conserved water molecule bridging two catalytic residues has been implicated. In human salivary alpha-amylase (HSAmy), this water (W641), observed in many alpha-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 2.1A revealed that the water chain is disrupted. In the F256W structure exhibits a positional shift in His305, characteristic of alpha-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 k(cat) (20-fold) and K(m) (4-fold) for maltoheptaoside. Collectively, these results suggest that W641 and the chain of water molecules may be critical for the alpha-amylase activity.

PubMed: 14678792
DOI: 10.1016/j.abb.2003.10.007

Experimental method

X-RAY DIFFRACTION (2.07 Å)