ISOZYME HYBRIDS WITHIN THE PROTRUDING 3RD LOOP DOMAIN OF THE BARLEY ALPHA-AMYLASE (BETA/ALPHA)(8)-BARREL IMPLICATION FOR BASI SENSITIVITY AND SUBSTRATE AFFINITY

Barley alpha-amylase isozymes AMY1 and AMY2 contain three structural domains: a catalytic (beta/alpha)(8)-barrel (domain A) with a protruding loop (domain B; residues 89-152) that binds Ca2+, and a small C-terminal domain. Different parts of domain B secure isozyme specific properties as identified for three AMY1-AMY2 hybrids, obtained by homeologous recombination in yeast, with crossing-over at residues 112, 116, and 144, The AMY1 regions Val(90)-Thr(112) and Ala(145)-Leu(161) thus confer high affinities for the substrates p-nitrophenyl alpha-D-maltoheptaoside and amylose, respectively. Leu(117)-Phe(144), and to a lesser degree Ala(145)-Leu(161), are critical for the stability at low pH characteristic of AMY1 and for the sensitivity to barley alpha-amylase/subtilisin inhibitor specific to AMY2.