SUBSITE MAPPING OF PORCINE PANCREATIC ALPHA-AMYLASE-I AND ALPHA-AMYLASE-II USING 4-NITROPHENYL-ALPHA-MALTOOLIGOSACCHARIDES

The catalytic efficiency (k(cat)/K-m) and the cleaved bond distribution for the nitrophenylated maltooligosaccharides, p-NPGlc(n) (2 less than or equal to n less than or equal to 7 ) hydrolysed by porcine pancreatic alpha-amylase isozymes I and II were determined. The subsite affinities (A,) were calculated from the p-NPGlc, (4 less than or equal to n less than or equal to 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.