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 d omains: a catalytic (beta/alpha)(8)-barrel (domain A) with a protrudin g loop (domain B; residues 89-152) that binds Ca2+, and a small C-term inal domain. Different parts of domain B secure isozyme specific prope rties as identified for three AMY1-AMY2 hybrids, obtained by homeologo us recombination in yeast, with crossing-over at residues 112, 116, an d 144, The AMY1 regions Val(90)-Thr(112) and Ala(145)-Leu(161) thus co nfer high affinities for the substrates p-nitrophenyl alpha-D-maltohep taoside and amylose, respectively. Leu(117)-Phe(144), and to a lesser degree Ala(145)-Leu(161), are critical for the stability at low pH cha racteristic of AMY1 and for the sensitivity to barley alpha-amylase/su btilisin inhibitor specific to AMY2.