a-amylase (a-1.4-glucan-4-glucanhydrolase) catalyzes the hydrolysis of a-(1,4) glycosidic linkages of starch components, glycogen, and various oligosaccharides. Furthermore, they are used in food and starch processing industry and, after proteases, have become the most used enzymes in modern biotechnology. The enzymatic properties of a-amylases from various species are rather complex, exhibiting different substrate specificities and product patterns. Most of the enzymatic studies have been carried out with porcine pancreatic a-amylase (PPA), which serves as a model system.
Porcine pancreatic a-amylase (PPA) is an endo-type amylase. It catalyzes the hydrolysis of internal a-(1,4) glucosidic bonds in amylose and amylopectin through multiple attack toward the nonreducing end. Two isoenzymes (PPAI and II) are known for pig pancreatic a-amylase. They have the same molecular weight, but differ slightly in amino acid composition and isoelectricity.
The three dimensional structures of a-amylases have now been reported from all major classes of organisms: from Aspergillus oryzae and A. niger, from porcine pancreas and human pancreas, from barley, and from Bacillus lichiformis. These studies have shown that the overall topology of a-amylase is extremely well conserved, despite the fact that their primary sequences can vary significantly. Less is known about the interactions of a-amylases with substrates and inhibitors. Details of the reaction mechanism for the hydrolysis of glycosidic linkages in substrates and the structural basis leading to different substrate specificities and product patterns remain to be solved.
Extensive screening has been carried out for inhibitors against various glucosidases in an attempt to develop therapeutics against diabetes, obesity, and hyperlipaemia. Two families have been studied in detail. One of these are the proteinaceous inhibitors discovered in bacteria of the genus Steptomyces. Recently the determination of the three-dimensional sructure of a complex of PPA with Tendamistat, from Streptomyces tendae, revealed interactions responsible for the very high dissociation constant of 9x10E11 M. The second set of inhibitors is derived from the so-called trestatin family, also found in Streptomyces. These pseudo-oligosaccharides contain acarviosine (an N-linked pseudo-dissacharide) and a varying number of glucose residues. In a recent study, native PPA crystals were soaked with trestatin, a derived pseudo-tetrasaccharide acarbose and the crystal structure determined at a resolution of 2.2 A. The final model showed a pseudo-pentasaccharide binding to the active site region indicating the PPA was able to process acarbose to amore potent inhibitor. Five sugar binding subsites could be located in the active site cleft. A further crystallographic soaking experiment with maltopentose resulted in the localization of two additional sugar binding sites (a glucose and a maltose) on the surface of PPA further apart from the active site region. The structure used to demonstrate the properties of PPA was derived from a cocrystallization of PPA and the trestin-derived pseudo-octasaccharide V-1532.