Barley malt-α-amylase. Purication, action pattern, and subsite mapping of isozyme 1 and two members of the isozyme 2 subfamily using p-nitrophenylated maltooligosaccharide substrates

Abstract

Isoform AMY1, AMY2-1 and AMY2-2 of barley α-amylase were purified from malt. AMY2-1 and AMY2-2 are both susceptible to barley α-amylase/subtilisin inhibitor. The action of these isoform is compared using substrates ranging from p-nitrophenylmaltoside through p-nitrophenylmaltoheptaoside. The k cat K m 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-nitrophynylmaltopentaoside, 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 k cat K m values show excellent agreement with experimental values.