Amylases structure

Structural analyses from X-ray models and predicted super-secondary models imply that CGTase structure is primarily a super-set of a-amylase structure. The active sites and calcium binding sites of a-amylase are believed to reside in the (p a)s barrel and P-strand loops of domains A and B. The various P-strand loops of the (p a)s barrel are also purported to be involved in starch binding. The antiparallel P-sheet (domain C) is hypothesized to possess starch binding capability. 

To this amylase structure, CGTase adds 2 other subdomains (D E), which are antiparallel P-sheets at the C-terminal end of the protein. The structure of domain D is similar to immimoglobulin topology. Tlie frmctional role of these domains is not known. However, proximity of domain E (an antiparallel P-sheet structure) to the proposed catalytic site, suggests a role in substrate binding and conformation. 

Matsuura, Y., et al. Structure and possible catalytic residues of taka-amylase A. /. Bioehem. 95 ... 

The stability of the enzyme-polymer complex and its dissociation upon the variation of pH depends on the structural and other physico-chemical properties of CP and enzyme molecule. Thus, a Biocarb-T heteroreticular biosorbent (Fig. 26) is characterized by a stability of its complex with ot-amylase (under the condition of its stabilization) in acid solutions and a complete dissociation of the complex during isolation of the active enzyme at pH 7-8. 

Several mono-carba-oligosaccharidic alpha amylase inhibitors, such as acarbose and its homologs, amylostatins, trestatins, oligostatins, adipo-sins, and so on, have been isolated from cultures of micro-organisms, and considerable interest in the biochemistry and chemistry of this class of inhibitors has been stimulated. The characteristic core-structure for inhibitory action is composed of a trihydroxy(hydroxymethyl)cyclohexene moiety and a 4-amino-4,6-dideoxy-D-glucopyranose moiety, bonded by way of an imino linkage at the allylic position. A similar structural unit has been found in the antibiotic validamycins. 

Crystal-structure analysis of Taka amylase A gave similar results, in that it showed that it had an extended cleft which could accommodate six, or possibly seven, a-( 1 — 4)-linked glucose units and two oppositely placed acidic amino acids (Asp-206 and Glu-230) which could interact with the bound substrate similarly to Asp-52 and Glu-35 in lysozyme. 

E. A. MacGregor, H. M. Jespersen, and B. Svensson, A circularly permuted alpha-amylase-type alpha/beta-barrel structure in glucan-synthesizing glucosyl-transferases, FEBS Lett., 378 (1996) 263-266. 

The only example of this technique applied to the amylose component is that already described, of the action of Z-enzyme on the /3-limit dextrin. In the case of amylopectin, enzymic methods enable a distinction to be made between the proposed laminated and highly ramified structures (I and III, in Fig. 1, page 352). The method used by Peat and coworkers101 involves the successive action of /3-amylase and R-enzyme on waxy maize starch. /3-Amylolysis will degrade A-chains down to two or three units from the 6 —> 1-a-D interchain linkages. These latter linkages will protect the... 

The enzymes in wheat, and hence in flour, that often cause problems in the bakery are present in the seed to make nutrient available to the seed. Similarly, this is why sprouted wheat causes problems if it is allowed to get into flour. Thus, the a-amylase is low in mature wheat grains but rises rapidly on germination. In bread, a low, but not too low, level of a-amylase is desirable since it produces sugars to feed the yeast and opens up the structure. Deliberate additions of malt flour were once common, but are now rarely made, to increase the amylase level. 

When Canadian wheat flour was the norm in British bakeries, with a falling number as high as 600, it was desirable to introduce malt flour to increase the a-amylase to feed the yeast and open up the structure of the crumb. 

The data19 summarized in Figure 1 show that the extent of the hydrolysis of soluble potato starch by barley beta amylase reaches a limit which is independent of the concentration of the amylase. The data are typical of the action of beta amylases on unfractionated starches, when the hydrolyses are carried out at or near pH 4.5.1 3 6 19 20 Under these conditions, the hydrolysis of unfractionated starches usually ceases when 60 to 64% of the maltose theoretically obtainable from the substrate has been formed. The exact value of the limit obviously will depend upon the concentration of amylopectin in the starch and upon its structure. 

If amylases are to be used as tools for the detailed study of the breakdown and structure of their substrates it is obviously important to separate them from other enzymes and from other naturally associated constituents which may influence the results. It is then equally important to study the properties of the purified amylase and to supply it with the chemical environment necessary to protect it from inactivation and to enable it to act efficiently. With beta amylases this ideal has often been approached. Beta amylases from several sources have been prepared by selective inactivation of other enzymes that accompany them in nature23 and highly active products have been obtained by extensive purification.20 24-26 Balls and his associates have recently reported the crystallization of beta amylase from sweet potato.27... 

Differences between the spectra of fluorescence and phosphorescence are immediately obvious. For all tryptophans in proteins the phosphorescence spectrum, even at room temperature, is structured, while the fluorescence emission is not. (Even at low temperatures the fluorescence emission spectrum is usually not structured. The notable exceptions include a-amylase and aldolase, 26 protease, azurin 27,28 and ribonuclease 7, staphylococcal endonuclease, elastase, tobacco mosaic virus coat protein, and Drosophila alcohol dehydrogenase 12. )... 

Affinity chromatography was carried out on columns prepared with lightly carboxymethylated chitin, which is known to be a poor substrate for lysozyme. Both native lysozyme and regenerated 13-105 were bound to the column at pH 7 and eluted at pH 3. As controls, the basic proteins cytochrome c and pancreatic RNase A, as well as concanavalin A and a-amylase, were not bound from the same solvent at pH 7. These findings constitute a third line of evidence for formation of native-like structure in regenerated 13-105. 

Structural aspects of proteinaceous cr -amylase inhibitor peptides 276... 

Structural aspects of proteinaceous a-amylase inhibitor peptides... 

To understand the inhibition of a-amylase by peptide inhibitors it is crucial to first understand the native substrate-enzyme interaction. The active site and the reaction mechanism of a-amylases have been identified from several X-ray structures of human and pig pancreatic amylases in complex with carbohydrate-based inhibitors. The structural aspects of proteinaceous a-amylase inhibition have been reviewed by Payan. The sequence, architecture, and structure of a-amylases from mammals and insects are fairly homologous and mechanistic insights from mammalian enzymes can be used to elucidate inhibitor function with respect to insect enzymes. The architecture of a-amylases comprises three domains. Domain A contains the residues responsible for catalytic activity. It complexes a calcium ion, which is essential to maintain the active structure of the enzyme and the presence of a chloride ion close to the active site is required for activation. 

The reaction mechanism of a-amylases is referred to as retaining, which means that the stereochemistry at the cleaved bond of the carbohydrate is retained. Hydrolysis of the glycosidic bond is mediated by an acid hydrolysis mechanism, which is in turn mediated by Aspl97 and Glu233 in pig pancreatic amylase. These interactions have been identified from X-ray crystallography. The aspartate residue has been shown to form a covalent bond with the Cl position of the substrate in X-ray structure of a complex formed by a structurally related glucosyltransferase. " The glutamate residue is located in vicinity to the chloride ion and acts as the acidic catalyst in the reaction. The catalytic site of a-amylases is located in a V-shaped depression on the surface of the enzyme. 

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