Journal
JOURNAL OF MOLECULAR STRUCTURE-THEOCHEM
Volume 666, Issue -, Pages 487-498Publisher
ELSEVIER SCIENCE BV
DOI: 10.1016/j.theochem.2003.08.073
Keywords
alpha-amylase; maltooligosaccharide; action pattern; subsite map; binding energy
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alpha-Amylases (alpha-1,4-glucan-4-glucanohydrolases; EC 3.2.1.1) are classical calcium-containing enzymes, which constitute a family of endo-amylases catalysing the cleavage of alpha-D-(1-4) glycosidic bonds in starch and related carbohydrates with retention of the alpha-anomeric configuration in the products. They can be found in microorganisms, plants and higher organisms where they play a dominant role in carbohydrate metabolism. This study characterizes the substrate binding sites of Bacillus licheniformis alpha-amylase (BLA), human salivary alpha-amylase (HSA) and its Y151M mutant. It describes the first subsite maps, namely, number of subsites, position of cleavage sites and apparent subsite energies. The product pattern and cleavage frequencies were determined by HPLC, utilising a homologous series of chromophore-substituted maltooligosaccharides of degree of polymerisation (DP) 3 - 10 as model substrates. 2-Chloro-4-nitrophenyl (CNP) and 4,6-O-benzylidene-modified 4-nitrophenyl (Bnl-NP) beta-maltooligosaccharides (DP 4-8) were synthesised from cyclodextrins using a chemical procedure. For the preparation of CNP-maltooligosides of longer chain length a new chemoenzymatic procedure was developed using rabbit skeletal muscle glycogen phosphorylase b. Our results confirmed the presence of eight binding sites in BLA, five glycone sites (-5, -4, -3, -2, - 1), three aglycone sites (+1, +2, +3) and the catalytic site is located between subsites (- 1 and + 1). In addition, the subsite map revealed a barrier site at the reducing end of active site which repulses the glucose residue. The binding region of HSA is composed of four glycone and three aglycone-binding sites, while that of Tyr151Met mutant is composed of four glycone and two aglycone-binding sites. The subsite maps show that Y151M has strikingly decreased binding energy at subsite (+ 2), where the mutation has occurred (- 2.6 kJ/mol), compared to the binding energy at subsite (+ 2) of HSA (- 12.0 kJ/mol). (C) 2003 Elsevier B.V. All rights reserved.
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