Porcine pancreatic alpha-amylase: a model for structure--function studies of homodepolymerases.

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.

Complete amino acid sequence and location of the five disulfide bridges in porcine pancreatic alpha-amylase.

Porcine pancreatic alpha-amylase (1,4-alpha-D-glucan glucanohydrolase EC 3.2.1.1), a single polypeptide chain, contains nine residues of methionine. Eight different fragments resulting from cleavage of this molecule by cyanogen bromide were characterized. The sequences of six of them have previously been reported. Two missing fragments, CN2 (82 residues) and CN3b1 (76 residues) were purified after breaking of the interpeptidic disulfide bridge and their complete sequence as well as that of the previously purified CN1 peptide (102 residues) are reported here. The location of the three disulfide bridges present in these peptides was determined. Ordering of the carboxymethylated cyanogen bromide fragments was carried out by pulse labeling the amylase chain in vivo. The complete sequence of the porcine pancreatic amylase chain (496 residues) and the location of its five disulfide bridges is presented. Comparison with human and mouse pancreatic and salivary alpha-amylases and with rat pancreatic amylase obtained from the corresponding cDNA nucleotidic sequences shows a high degree of homology between mammalian alpha-amylases.