Subsite mapping of porcine pancreatic alpha-amylase I and II using 4-nitrophenyl-alpha-maltooligosaccharides.
The catalytic efficiency (kcat/Km) and the cleaved bond distribution for the nitrophenylated maltooligosaccharides, p-NPGlcn (2 < or = n < or = 7) hydrolysed by porcine pancreatic alpha-amylase isozymes I and II were determined. The subsite affinities (Ai) were calculated from the p-NPGlcn (4 < or = n < or = 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.
Ajandouz
E H
EH
Laboratoire de Biochimie et Biologie de la Nutrition, URA-CNRS 1820, Université d'Aix-Marseille III, Faculté des Sciences, France.
Marchis-Mouren
G J
GJ
D000818:Animals; D005960:Glucosides / Q000737:chemistry*; D007527:Isoenzymes / Q000737:chemistry*; D007700:Kinetics; D009578:Nitrobenzenes / Q000737:chemistry; D009844:Oligosaccharides / Q000737:chemistry; D010179:Pancreas / Q000201:enzymology*; D013379:Substrate Specificity; D013552:Swine; D000516:alpha-Amylases / Q000737:chemistry*
D005960:Glucosides; D007527:Isoenzymes; D009578:Nitrobenzenes; D009844:Oligosaccharides; C037063:4-nitrophenyl-alpha-maltotetraose; C075379:4-nitrophenyl maltopentaoside; C036167:4-nitrophenyl maltoheptaoside; D000516:alpha-Amylases