Site-directed mutagenesis of histidine 93, aspartic acid 180, glutamic acid 205, histidine 290, and aspartic acid 291 at the active site and tryptophan 279 at the raw starch binding site in barley alpha-amylase 1.
The pseudotetrasaccharide acarbose has high affinity for the active site (Ki,app = 1 microM) and low affinity for a secondary site (Kd = 2.3 mM) in barley alpha-amylase 1, distinguished by inhibition kinetics and spectral perturbation. Mutants of putative catalytic residues, D180N, E205Q, and D291N, are inactive and display low affinity for acarbose-Sepharose. H93N and H290N mutants, at invariant residues, have kcat/Km for p-nitrophenylmaltoheptaoside of 0.3 and 1.2% of wild-type. A corresponding 370- and 85-fold increased Ki,app for acarbose and a lack of shifts in pH activity profiles indicate that these histidines participate in transition state stabilization but not directly in catalysis. This finding agrees with H bonding to OH groups of the valienamine ring of acarbose in the three-dimensional structure. Loss of inhibition above pH 6 supports that acarbose is most potent in protonated form. The low affinity site contains Trp278 and Trp279, known to bind cyclomaltoheptaose. While the W279A mutant has 10-fold decreased affinity for starch granules, production of Trp278 mutants failed. The invariant Trp278 is perhaps critical for stability or folding in cereal alpha-amylases.
Søgaard
M
M
Carlsberg Laboratory, Department of Chemistry, Valby, Denmark.
Kadziola
A
A
Haser
R
R
Svensson
B
B
D000595:Amino Acid Sequence; D001224:Aspartic Acid; D001483:Base Sequence; D001665:Binding Sites; D003001:Cloning, Molecular; D017931:DNA Primers; D005971:Glutamates; D018698:Glutamic Acid; D006639:Histidine; D001467:Hordeum / Q000201:enzymology*; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012441:Saccharomyces cerevisiae; D014364:Tryptophan; D000516:alpha-Amylases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
D017931:DNA Primers; D005971:Glutamates; D011994:Recombinant Proteins; D001224:Aspartic Acid; D018698:Glutamic Acid; D006639:Histidine; D014364:Tryptophan; D000516:alpha-Amylases