Involvement of individual subsites and secondary substrate binding sites in multiple attack on amylose by barley alpha-amylase.
Barley alpha-amylase 1 (AMY1) hydrolyzed amylose with a degree of multiple attack (DMA) of 1.9; that is, on average, 2.9 glycoside bonds are cleaved per productive enzyme-substrate encounter. Six AMY1 mutants, spanning the substrate binding cleft from subsites -6 to +4, and a fusion protein, AMY1-SBD, of AMY1 and the starch binding domain (SBD) of Aspergillus niger glucoamylase were also analyzed. DMA of the subsite -6 mutant Y105A and AMY1-SBD increased to 3.3 and 3.0, respectively. M53E, M298S, and T212W at subsites -2, +1/+2, and +4, respectively, and the double mutant Y105A/T212W had decreased DMA of 1.0-1.4. C95A (subsite -5) had a DMA similar to that of wild type. Maltoheptaose (G7) was always the major initial oligosaccharide product. Wild-type and the subsite mutants released G6 at 27-40%, G8 at 60-70%, G9 at 39-48%, and G10 at 33-44% of the G7 rate, whereas AMY1-SBD more efficiently produced G8, G9, and G10 at rates similar to, 66%, and 60% of G7, respectively. In contrast, the shorter products appeared with large individual differences: G1, 0-15%; G2, 8-43%; G3, 0-22%; and G4, 0-11% of the G7 rate. G5 was always a minor product. Multiple attack thus involves both longer translocation of substrate in the binding cleft upon the initial cleavage to produce G6-G10, essentially independent of subsite mutations, and short-distance moves resulting in individually very different rates of release of G1-G4. Accordingly, the degree of multiple attack as well as the profile of products can be manipulated by structural changes in the active site or by introduction of extra substrate binding sites.
Kramhøft
Birte
B
Department of Chemistry, Carlsberg Laboratory, Gamle Carlsberg Vej 10, DK-2500 Copenhagen Valby, Denmark.
Bak-Jensen
Kristian Sass
KS
Mori
Haruhide
H
Juge
Nathalie
N
Nøhr
Jane
J
Svensson
Birte
B
D000688:Amylose / Q000737:chemistry* / Q000378:metabolism; D001665:Binding Sites / Q000235:genetics; D014644:Genetic Variation; D005936:Glucans / Q000378:metabolism; D005947:Glucose / Q000378:metabolism; D001467:Hordeum / Q000201:enzymology* / Q000235:genetics; D006868:Hydrolysis; D007527:Isoenzymes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D008320:Maltose / Q000031:analogs & derivatives / Q000378:metabolism; D009844:Oligosaccharides / Q000737:chemistry / Q000378:metabolism; D010940:Plant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity / Q000235:genetics; D014312:Trisaccharides / Q000378:metabolism; D000516:alpha-Amylases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
D005936:Glucans; D007527:Isoenzymes; D009844:Oligosaccharides; D010940:Plant Proteins; D014312:Trisaccharides; C019193:maltopentaose; C016549:maltohexaose; C031146:maltoheptaose; C008317:maltotriose; D008320:Maltose; D000688:Amylose; C009819:maltotetraose; C113880:AMY1.2 protein, Hordeum vulgare; D000516:alpha-Amylases; D005947:Glucose