The 'pair of sugar tongs' site on the non-catalytic domain C of barley alpha-amylase participates in substrate binding and activity.
Some starch-degrading enzymes accommodate carbohydrates at sites situated at a certain distance from the active site. In the crystal structure of barley alpha-amylase 1, oligosaccharide is thus bound to the 'sugar tongs' site. This site on the non-catalytic domain C in the C-terminal part of the molecule contains a key residue, Tyr380, which has numerous contacts with the oligosaccharide. The mutant enzymes Y380A and Y380M failed to bind to beta-cyclodextrin-Sepharose, a starch-mimic resin used for alpha-amylase affinity purification. The K(d) for beta-cyclodextrin binding to Y380A and Y380M was 1.4 mm compared to 0.20-0.25 mm for the wild-type, S378P and S378T enzymes. The substitution in the S378P enzyme mimics Pro376 in the barley alpha-amylase 2 isozyme, which in spite of its conserved Tyr378 did not bind oligosaccharide at the 'sugar tongs' in the structure. Crystal structures of both wild-type and S378P enzymes, but not the Y380A enzyme, showed binding of the pseudotetrasaccharide acarbose at the 'sugar tongs' site. The 'sugar tongs' site also contributed importantly to the adsorption to starch granules, as Kd = 0.47 mg.mL(-1) for the wild-type enzyme increased to 5.9 mg.mL(-1) for Y380A, which moreover catalyzed the release of soluble oligosaccharides from starch granules with only 10% of the wild-type activity. beta-cyclodextrin both inhibited binding to and suppressed activity on starch granules for wild-type and S378P enzymes, but did not affect these properties of Y380A, reflecting the functional role of Tyr380. In addition, the Y380A enzyme hydrolyzed amylose with reduced multiple attack, emphasizing that the 'sugar tongs' participates in multivalent binding of polysaccharide substrates.
Bozonnet
Sophie
S
Enzyme and Protein Chemistry, BioCentrum-DTU, Technical University of Denmark.
Jensen
Morten T
MT
Nielsen
Morten M
MM
Aghajari
Nushin
N
Jensen
Malene H
MH
Kramhøft
Birte
B
Willemoës
Martin
M
Tranier
Samuel
S
Haser
Richard
R
Svensson
Birte
B
D000595:Amino Acid Sequence; D001483:Base Sequence; D020134:Catalytic Domain; D018360:Crystallography, X-Ray; D017931:DNA Primers; D001467:Hordeum / Q000201:enzymology*; D006868:Hydrolysis; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D020349:Surface Plasmon Resonance; D000516:alpha-Amylases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D047392:beta-Cyclodextrins / Q000378:metabolism