Amylases active sites

Fig. 1. Inhibition of porcine pancreatic a-amylase. Substrates, an inhibitor, and their binding orientations in the active site are shown schematically. The arrows denote the catalytic site in each case, (a) The small substrate, G2PNP [17400-77-0] (3) (b) the large substrate, G OH [13532-61 -1] (4) and (c) the inhibitor, 4-phenyl imidazole (5) and the substrate G2PNP (3) in the binding orientation for noncompetitive inhibition. The binding orientation of G2PNP...

MIRKOV T E, EVANS S V, WAHLSTROM J, GOMEZ L, YOUNG N M, CHRISPEELS M J (1995) Location of the active site of the bean alpha-amylase inhibitor and involvement of a Trp, Arg, Tyr triad. Glycobiology. 5 45-50. 

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. 

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. 

The interactions of a-amylases, mainly porcine pancreatic a-amylase, and thiomaltodextrins have been investigated. In 1980, the 3-D structure of porcine pancreatic a-amylase was reported [68], and an analogue of the thiomdtotrio-side (48b), prepared by standard condensation between (34e) and (51c), was effective to label the active site and to identify a second binding site on the siu -face of the protein molecule. 

Figure 12-6 Drawing showing the overall polypeptide chain fold and relative positioning of the three structural domains of human pancreatic a-amylase. Also drawn are the locations of the calcium and chloride binding sites. Overlaid is the placement of a modified form of the inhibitor acarbose (p. 607) that binds in the active site cleft. MolScript drawing courtesy of G. Sidhu and G. Brayer.

Once in the active site, the substrate undergoes reaction. The product is then released for use in the next stage, which is controlled by another enzyme, and the original enzyme molecule is free to receive the next substrate molecule. One example is the enzyme amylase present in your mouth. The amylase in saliva helps to break down starches in food into the more easily digested glucose. If you chew a cracker long enough, you can notice the increased sweetness. 

Modification reactions that neutralize charges or introduce hydro-phobic residues usually lower the enzymic activity. The attachment of monosaccharides to alpha amylase by diazo coupling lowered the activity.12 This enzyme was stable to the reaction conditions for diazo coupling (pH 10,15 min at 0°) if the diazonium salts were not included in the solution. Inclusion of maltose in the reaction mixture to protect the active site lessened, but did not eliminate, the loss of activity, suggesting that the incorporation of hydrophobic structures, or the modification of a critical residue distant from the active site, was at least partly responsible for the loss of activity. 

For a hormone to have a specific effect on gene activity, any increase in enzyme activity must result from de novo synthesis by newly formed mRNA. This increase in enzyme activity may or may not precede any general increase in metabolic activity. From the foregoing discussion on chromatin activity, it is clear that plant hormones largely either increase the activity of polymerase I or increase the synthesis of total RNA s. Claims that the hormones "activate" chromatin-bound polymerases and "modulate" the number of active sites on the chromatin (21) have not been substantiated. There are only two known examples of hormone-induced synthesis of specific mRNA s. The classic example is the barley aleurone cells, in which GA treatment induces de novo synthesis and release of K-amylase (58, 59, 60), protease (61), and possibly as many as ten proteins (62). 

A number of starch-converting enzymes belong to a single family termed the a-amylase family or family 13 hydrolases. This group of enzymes shares common characteristics such as an eight-stranded a/p barrel structure, the ability to hydrolyze 1,4-a-D-glucosidic linkages of attached polysaccharides in a-conformation, and conserved amino acid residues in the active sites of the enzymes (van der Maarel et al. 2002). 

Figure 7.9 Topological structures of a-amylase A. Two-dimensional representation of the secondary and domain structures of porcine pancreatic a-amylase. Alpha helices are represented as circles and (3-strands in the up-direction as squares, and in the down direction as double squares. The (a/(3)g—TIM barrel comprises domain A. Hydrogen bonds between (3-strands are shown by dashed lines. The a-helices and (3-strands are identified in the various domains by A, B and C. (Reprinted by permission ofthe authors M. Qian et al.120) Two-dimensional representation ofthe secondary and domain structures of barley malt a-amylase (AMY2-2). Alpha helices are represented as cylinders and (3-strands as arrows. The (a/(3)g—TIM barrel comprises domain A, with eight (3-strands and an equivalent of eight a-helices. The active-site is composed ofthe loops that connect the C-termini ofthe (3-strands to the N-termini ofthe peripheral a-helices. (Adapted from A. Kadziola et al.121)...

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