Two additional carbohydrate-binding sites of beta-amylase from Bacillus cereus var. mycoides are involved in hydrolysis and raw starch-binding.
In the previous X-ray crystallographic study, it was found that beta-amylase from Bacillus cereus var. mycoides has three carbohydrate-binding sites aside from the active site: two (Site2 and Site3) in domain B and one (Site1) in domain C. To investigate the roles of these sites in the catalytic reaction and raw starch-binding, Site1 and Site2 were mutated. From analyses of the raw starch-binding of wild-type and mutant enzymes, it was found that Site1 contributes to the binding affinity to raw-starch more than Site2, and that the binding capacity is maintained when either Site1 or Site2 exists. The raw starch-digesting ability of this enzyme was poor. From inhibition studies by maltitol, GGX and alpha-CD for hydrolyses of maltopentaose (G5) and amylose ( (n) = 16) catalyzed by wild-type and mutant enzymes, it was found that alpha-CD is a competitive inhibitor, while, maltitol behaves as a mixed-type or competitive inhibitor depending on the chain length of the substrate and the mutant enzyme. From the analysis of the inhibition mechanism, we conclude that the bindings of maltitol and GGX to Site2 in domain B form an abortive ESI complex when amylose ( (n) = 16) is used as a substrate.
Ye
Zhengmao
Z
Laboratory of Enzyme Chemistry, Graduate School of Agriculture and Biological Science, Osaka Prefecture University, Sakai, Osaka 599-8531, Japan.
Miyake
Hideo
H
Tatsumi
Maki
M
Nishimura
Shigenori
S
Nitta
Yasunori
Y
D001409:Bacillus cereus / Q000201:enzymology* / Q000235:genetics; D001665:Binding Sites; D001667:Binding, Competitive; D006868:Hydrolysis / Q000187:drug effects; D007700:Kinetics; D008320:Maltose / Q000031:analogs & derivatives* / Q000378:metabolism / Q000494:pharmacology; D008958:Models, Molecular; D009154:Mutation / Q000235:genetics; D009844:Oligosaccharides / Q000378:metabolism; D011485:Protein Binding; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013057:Spectrum Analysis; D013213:Starch / Q000378:metabolism*; D013402:Sugar Alcohols / Q000378:metabolism / Q000494:pharmacology; D047391:alpha-Cyclodextrins / Q000378:metabolism / Q000494:pharmacology; D001614:beta-Amylase / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
D009844:Oligosaccharides; D011994:Recombinant Proteins; D013402:Sugar Alcohols; D047391:alpha-Cyclodextrins; C019193:maltopentaose; D008320:Maltose; D013213:Starch; C010745:maltitol; D001614:beta-Amylase; C032613:alpha-cyclodextrin