Subsite mapping of porcine pancreatic alpha-amylase I and II using 4-nitrophenyl-α-maltooligosaccharides

The catalytic efficiency (kcat/Km) and the cleaved bond distribution for the nitrophenylated maltooligosaccharides, p-NPGlcn (2 ⩽ n ⩽ 7) hydrolysed by porcine pancreatic alpha-amylase isozymes I and II were determined. The subsite affinities (Ai) were calculated from the p-NPGlcn (4 ⩽ n ⩽ 7) hydrolysis data. Five subsites (−3 to 2) bind glucosidic residues with a positive affinity. No additional subsites could be detected both at the reducing end (3, 4, 5) and at the nonreducing end (−4, −5, −6). The energetic profiles of both isozymes are similar. The energetic profile of PPA differs from other alpha-amylases by having both a small number of subsites, and a catalytic subsite with a high positive affinity. Excellent agreement was found between observed catalytic efficiency values and those calculated from the subsite affinities.