structure and reacting groups on amylase

[sitarampharma]

Hi everybody............
I am sita ram, i am working on amylase and as a part of that i want to know the reacting groups on amylase and also the binding capacity of different metals to that enzyme.

[Zagami]

From my work “Zagami F. (2002) 2-Chloro-4-nitrophenyl-ß-D-maltotrioside, new substrate for α-amilase determination in biological fluids. Int. J. Clin. Invest. Vol. X, n°. 1, March, 39- 43” amylase is mainly involved in the process of digestion since it catalyses the hydrolysis of α-1,4 glucan linkages in amyloses (e.g. starch) to produce maltose and glucose. In humans amylase is composed about 496 amino acids in a single polypeptide chain, one activator chloride ion, one structural calcium ion, and 393 water molecules, encoded on chromosome 1 as part of a multigene family. In the cleft of the active site a catalytic trio of acidic groups Asp197, Glu233 and Asp300, is located. Glu233 and Asp300 suggests their role in acid/base catalysis in the reaction mechanism. Glu233 is believed to be the catalyst since its pKa is increased with respect to Asp300 because of its close location to the chloride ion. Maximal enzymatic activity occurs at approximately pH 7. By the confluence of catalytic triad the final active site is represented by a carboxyl group of aspartic acid/glutamic acid. Formation of the intermediate involves attack at the sugar anomeric center by a nucleophilic amino acid (Asp197), assisted by general-acid catalysis (Glu233). The chloride ion bound in the direct vicinity of the active site and may serve as an allosteric activator of catalysis (enzymatic activation). Chloride ion coordination involves the side chains, chloride ion-binding site, Arg195, Asn298, and Arg 337, residues located in direct vicinity of the active site, and form a hydrogen bond, along with a water molecule, network involving the catalytic residues Asp197 and Asp300. Chloride is required to increase the pKa of the acid/base catalyst, Glu233, which would otherwise be lower due to the nearby presence of Arg337 (a positively charged residue). The calcium ion binding pocket is essential to the stability of the polypeptide chain fold. Calcium does not play a direct part in catalysis, but (along with the chloride ion) prepares a proper geometric conformation of the active site. The key residue is Asn100, which is hydrogen-bonded to the catalytic Asp197 (which in turn interacts with the chloride ion). Amylase is usually found in two forms : α-amylase and β-amylase. They have a similar function but work in a different way. In the human body, α-amylase is found in saliva and pancreatic juices but they are not completely identical, and β-amylase is found in stomach juices and works at a different place on sugars compared with α-amylase.