Models for the action of barley alpha-amylase isozymes on linear substrates

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

“…(Table I). In contrast, the conservative Y105(F/W) mutants resembled wildtype AMY1 closely, Y105F and Y105W being slightly superior on starch and on both Cl-PNPG 7 and amylose DP17, respectively. The loss of the substrate binding ␥OH group in Y105F decreased affinity (3-fold) only for Cl-PNPG 7 (Table I) (Table I), increasing the affinity 5-and 2-fold and k cat /K m to 370% of T212Y and 180% of T212W compared with wild type.…”

“…Effects of Mutations on Oligosaccharide Action PatternsMutation at specific subsites can reposition short substrates in the binding cleft resulting in product profile engineering (39 -41, 66 7 and less PNPG, which is the predominant wild-type product, thus reflecting relative gain and loss of aglycone and glycone binding, respectively (Fig. 2).…”

“…(Table II) is also functionally important, and the AMY2 mimic D97E AMY1 reduced affinity for Cl-PNPG 7 and amylose DP17 4-and 2-fold, respectively (43).…”

“…Considering T212Y and T212W as parents of the double mutants, ⌬⌬G was 2.3 and 1.4 kcal ϫ mol Ϫ1 , respectively. In relation to Cl-PNPG 7 an estimate of (k cat /K m ) mutant / (k cat /K m ) parent Ͼ 100 for Y105A gave to ⌬⌬G Ͼ 2.8 kcal ϫ mol Ϫ1 and using T212Y and T212W as parents both gave ⌬⌬G ϭ 1.6 kcal ϫ mol Ϫ1 . These losses are smaller than the calculated energy of 10 kcal ϫ mol Ϫ1 corresponding to loss of stacking for Y105A in S2 AMY1 at subsite Ϫ6 (Table II A) or than the difference between S1 AMY1 and S2 AMY1 of 34 kcal ϫ mol Ϫ1 .…”

“…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).…”