Structural studies of a Phe256Trp mutant of human salivary alpha-amylase: implications for the role of a conserved water molecule in enzyme activity.
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.
Ramasubbu
Narayanan
N
Department of Oral Biology, New Jersey Dental School, University of Medicine and Dentistry, Newark, NJ 07103, USA. n.ramasubbu@umdnj.edu
Sundar
Krishnan
K
Ragunath
Chandran
C
Rafi
Mohamed M
MM
D019943:Amino Acid Substitution; D001665:Binding Sites; D002384:Catalysis; D018360:Crystallography, X-Ray; D006801:Humans; D006868:Hydrolysis; D007700:Kinetics; D015394:Molecular Structure; D016297:Mutagenesis, Site-Directed; D009844:Oligosaccharides / Q000737:chemistry / Q000378:metabolism; D010649:Phenylalanine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013213:Starch / Q000737:chemistry / Q000378:metabolism; D014364:Tryptophan / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D014867:Water / Q000737:chemistry* / Q000378:metabolism*; D000516:alpha-Amylases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
D009844:Oligosaccharides; D011994:Recombinant Proteins; D014867:Water; D010649:Phenylalanine; D014364:Tryptophan; D013213:Starch; D000516:alpha-Amylases