Barley malt-alpha-amylase. Purification, action pattern, and subsite mapping of isozyme 1 and two members of the isozyme 2 subfamily using p-nitrophenylated maltooligosaccharide substrates.
Isoforms AMY1, AMY2-1 and AMY2-2 of barley alpha-amylase were purified from malt. AMY2-1 and AMY2-2 are both susceptible to barley alpha-amylase/subtilisin inhibitor. The action of these isoforms is compared using substrates ranging from p-nitrophenylmaltoside through p-nitrophenylmaltoheptaoside. The kcat/Km values are calculated from the substrate consumption. The relative cleavage frequency of different substrate bonds is given by the product distribution. AMY2-1 is 3-8-fold more active than AMY1 toward p-nitrophenylmaltotrioside through p-nitrophenylmaltopentaoside. AMY2-2 is 10-50% more active than AMY2-1. The individual subsite affinities are obtained from these data. The resulting subsite maps of the isoforms are quite similar. They comprise four and six glucosyl-binding subsites towards the reducing and the non-reducing end, respectively. Towards the non-reducing end, the sixth and second subsites have a high affinity, the third has very low or even lack of affinity and the first (catalytic subsite) has a large negative affinity. The affinity declines from moderate to low for subsites 1 through 4 toward the reducing end. AMY1 has clearly a more negative affinity at the catalytic subsite, but larger affinities at both the fourth subsites, compared to AMY2. AMY2-1 has lower affinity than AMY2-2 at subsites adjacent to the catalytic site, and otherwise mostly higher affinities than AMY2-2. Theoretical kcat/Km values show excellent agreement with experimental values.
Ajandouz
E H
EH
BBMN Faculté des Sciences, Université d'Aix-Marseille III, France.
Abe
J
J
Svensson
B
B
Marchis-Mouren
G
G
D004591:Electrophoresis, Polyacrylamide Gel; D005947:Glucose / Q000378:metabolism; D005960:Glucosides / Q000378:metabolism*; D001467:Hordeum / Q000201:enzymology*; D007525:Isoelectric Focusing; D007527:Isoenzymes / Q000302:isolation & purification* / Q000378:metabolism; D009844:Oligosaccharides / Q000378:metabolism*; D013379:Substrate Specificity; D000516:alpha-Amylases / Q000037:antagonists & inhibitors / Q000302:isolation & purification* / Q000378:metabolism
D005960:Glucosides; D007527:Isoenzymes; D009844:Oligosaccharides; C021705:maltooligosaccharides; C036988:4-nitrophenylmaltoside; D000516:alpha-Amylases; D005947:Glucose