title: Porcine pancreatic alpha-amylase: a model for structure--function studies of homodepolymerases.
abstract: The amino acid sequence of the porcine pancreatic alpha-amylase chain (496 residues) contains four regions (96-101, 193-201, 233-236 and 295-300) which are highly homologous in amylases of different origins. These regions all belong to the N-terminal domain of the enzyme. Limited proteolysis by subtilisin allows a cut to be made at bond 369-370. Purified fragments indicate that both N- and C-terminal domains are required for amylolytic activity. Kinetic studies and reaction product analysis using oligomaltosides, their nitrophenylated derivatives and amylose as the substrate allowed us to establish: 1) the energy profile of the 5 subsites and, especially, that subsite number 3 is catalytic; 2) that 2 molecules of either maltotriose or its o-nitrophenylated analog or maltose bind to the active site at high substrate concentration. Such a subsite occupancy was confirmed by fluorescence quenching studies. Finally the hydrolysis of p-nitrophenylmaltoside was studied as a function of pH. In contrast to starch hydrolysis, the initial velocity plots for nitrophenol and p-nitrophenylglucoside liberation both gave a narrow pH-activity peak with a maximum value around pH 5.5. All data provide strong evidence for the participation of 2 carboxylic residues in the catalysis.
authors.affiliation: Institut de Chimie Biologique, Université d'Aix-Marseille, France.
authors.forename: V
authors.initials: V
authors.lastname: Desseaux
authors.forename: C
authors.initials: C
authors.lastname: Seigner
authors.forename: Y
authors.initials: Y
authors.lastname: Pierron
authors.forename: M L
authors.initials: ML
authors.lastname: Grisoni
authors.forename: G
authors.initials: G
authors.lastname: Marchis-Mouren
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D006868:Hydrolysis; D007700:Kinetics; D010179:Pancreas / Q000201:enzymology*; D011487:Protein Conformation; D013329:Structure-Activity Relationship; D013552:Swine; D000516:alpha-Amylases / Q000378:metabolism*
chemical_list: D000516:alpha-Amylases