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

Biochim Biophys Acta. 1992 Sep 23;1159(2):193-202. doi: 10.1016/0167-4838(92)90025-9.


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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Electrophoresis, Polyacrylamide Gel
  • Glucose / metabolism
  • Glucosides / metabolism*
  • Hordeum / enzymology*
  • Isoelectric Focusing
  • Isoenzymes / isolation & purification*
  • Isoenzymes / metabolism
  • Oligosaccharides / metabolism*
  • Substrate Specificity
  • alpha-Amylases / antagonists & inhibitors
  • alpha-Amylases / isolation & purification*
  • alpha-Amylases / metabolism


  • Glucosides
  • Isoenzymes
  • Oligosaccharides
  • maltooligosaccharides
  • 4-nitrophenylmaltoside
  • alpha-Amylases
  • Glucose