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

Improved Activity and Modulated Action Pattern Obtained by Random Mutagenesis at the Fourth β-α Loop Involved in Substrate Binding to the Catalytic (β/α)8-Barrel Domain of Barley α-Amylase 1

“…In no case, however, did the action pattern shift on G 5 -PNP, although SGM-AMY1 generated an unusually large amount of glucose (Table II). The results reflect the subsite map of AMY1 (19) with its low binding affinity at subsites ϩ3 and Ϫ3 and the high affinity at subsite Ϫ6 that plays an important role in binding near the nonreducing end of longer maltooligosaccharides.…”

“…Restriction enzymes were from Promega and were used according to the manufacturer's recommendation. AMY2 was purified from barley malt (cultivars Triumph or Menuet) (19,40) and BASI from barley seeds (cultivar Piggy) (41). Rabbit antibodies against barley ␣-amylase were raised using AMY2 as antigen (custom immunization by Dako A/S, Denmark).…”

Tyrosine 105 and Threonine 212 at Outermost Substrate Binding Subsites –6 and +4 Control Substrate Specificity, Oligosaccharide Cleavage Patterns, and Multiple Binding Modes of Barley α-Amylase 1

“…The subsite map of both isozymes contains 10 consecutive subsites of varying affinity, i.e. subsites Ϫ6 through Ϫ1 from the catalytic site toward the non-reducing and subsites ϩ1 through ϩ4 toward the reducing end of the substrate (5). Consistent with this subsite organization barley ␣-amylases release mainly oligosaccharides of DP 6Ϫ8 from starch (5-7), whereas most other ␣-amylases give shorter products (1,2,44).…”

“…A maltodecaose complex was computed earlier by stepwise addition of glucosyl residues extending the acarbose molecule bound in AMY2 (10) beyond subsites Ϫ1 and ϩ2. This complex revealed that Tyr 104 AMY2 and Tyr 211 AMY2 delimit the binding groove at subsites Ϫ6 and ϩ4 (5,25,46,47). The present incorporation and removal of aromatic residues at these positions in AMY1 thus manipulate common features in protein-carbohydrate interactions, i.e.…”

“…Substrate interactions along the extended binding site have traditionally been described by subsite maps that indicate the number of consecutive glucosyl binding subsites (ranging from [5][6][7][8][9][10][11], the cleavage position, and the affinity of substrate glucosyl residues at individual subsites (2)(3)(4)(5)(6)(7)(8). The binding cleft is formed by ␤ 3 ␣ loops of the catalytic (␤/␣) 8 barrel domain (9 -14).…”