Created by Sara Schutte
Salivary alpha-amylase belongs to the glycoside hydrolase family, which is an important family of proteins that is characterized by the ability to catalyze the hydrolysis of alpha-1,4-glucosidic bonds (Ragunath et al., 2008). Evidence indicates that salivary alpha-amylase is a vital first step in the digestion process in humans because of its ability to bind and break down starch and other polysaccharides (Ramasubbu et al., 1996).
Salivary alpha amylase consists of one subunit; a polypeptide chain of 496 amino acids, which forms three distinct domains A, B, and C. "Restriction mapping and nucleotide (nt) sequence analysis revealed that this gene is approximately 10 kb long and is separated into eleven exons by ten introns. Its 5'-flanking region has some sequence homology with that of mouse salivary alpha-amylase gene"(Nishide et al., 1986). According to Ragunath et al. (2008, p.3) it is likely that the ability of salivary alpha-amylase to bind starch (as well as bacteria) arises from the specific location of aromatic residues, including Tryptophan 58 and 59, which contribute potential stacking interactions at binding sites.
Salivary alpha-amylase is also a calcium binding protein, which requires that a calcium ion be bound in order to make the protein functional (Ramasubbu et al., 1996). A significant function of salivary alpha-amylase is that in the absence of human pancreatic alpha amylase, which is the primary enzyme for starch digestion, salivary alpha-amylase "may represent a potential compensatory alternate pathway for the digestion of amylose, amylopectin, and glycogen" (Lebenthal, 1987). This becomes especially vital in diseases like chronic pancreatitis and cystic fibrosis, and also during the first six months of an infant's life, in which the level of pancreatic alpha-amylase is "very low or absent" (Lebenthal, 1987). In cases such as this, salivary amylase is critical not only for the breakdown of starch but also for the maintainence of oral health. As noted by Ramasubbu et al. (1996, p. 435), salivary amylase is the most abundant enzyme in human saliva, most likely because it has at least three distinct functions: hydrolytic activity when breaking down starch, binding oral bacteria, and binding to the hydroxyapatite of the teeth, which plays a role in the formation of plaque on teeth.
Ragunath et al. (2008, p.2) also assert that the molecular structure of salivary alpha-amylase is very similar to other mammalian alpha-amylases, such as human pancreatic alpha-amylase. The molecular structure of adapted by salivary alpha-amylase is very similar to human pancreatic alpha-amylase, which could be because the sequence identity between the two proteins is greater than 97% (Ragunath et al., 2008).
However, there exist certain differences between the amylase found in saliva and the one found in pancreatic secretions. For example, salivary amylase is controlled by the Amy1 locus of the chromosome whereas pancreatic amylase is controlled by the Amy2 locus. They can also be separated by ion-exchange chromatography or polyacrylamide gel electrophoresis, which indicates other significant structural and compositional differences between the two proteins (Lebenthal 1987). In addition, human salivary alpha-amylase is inhibited "by wheat seed (Triticum aestivum), a type II alpha-amylase inhibitor, and it was revealed that the inhibition was slow and tight-binding" (Goff 1995). All of the biological activities of salivary alpha amylase "seem to depend on an intact enzyme conformation" (Scannapieco et al., 1993). A few of the most important residues in the salivary alpha-amylase molecule are the histidine (His-201) and tryptophan (Trp-58 and Trp-59) residues in the active site. These residues "may play a role in differentiating between the glycine and glycone ends of the polysaccharide substrates" (Ramasubbu et al., 1996). Since starch digestion is one of the most important functions of salivary alpha-amylase, these residues are essential to the function and significance of the protein.
Salivary alpha-amylase is an enzyme that is folded into three distinct domains A, B, and C. It is glycosylated and also binds a calcium ion and a chloride ion in order to function properly. It is considered a hydrolase and 444 water molecules are typically found coordinated to the molecule in its natrual state (Ramasubbu et al., 1996). Domain A is found in residues 1-99 and 169-404. This is the central domain of the protein, and it exhibits a barrel structure. The catalytic binding site for polysaccharides is found in domain A with the catalytic residuesAsp-197, Glu-233, and Asp-300. These residues are clustered next to each other in the binding cleft. Asp-197 also carries a formal charge. There are also seven to nine aromatic residues, including Trp-58 and Trp-59, that could exist in the cleft for glucose binding. The side chain of Trp-59 is found in a localized conformation when the protein is un-liganded but when a ligand is bound, the Trp-59 undergoes a conformational change, hydrogen bonding to the penultimate glucose residue of the saccharide bound. The substrate binding cleft in salivary alpha-amylase can differentiate between the reducing and non-reducing ends of substrates because of the Trp-58 and Trp-59 residues, which enter into hydrophobic and stacking interactions with the reducing end of the substrate. In addition, the N-terminus of salivary amylase is found in the A domain, and the acidic residues on this end adopt a flat conformation that allows the protein to bind to hydroxyapatite (Ramasubbu et al., 1996). Domain A is also where the chloride ion binds, which is necessary in order to neutralize the basic residues in the substrate binding cleft. One of the most important parts of the protein is the flexible loop region from residues 304 to 310, which is integral to the hydrolysis of saccharides. A large number of glycine residues are present in this area, which increases the flexibility of the loop. When a substrate is bound the loop becomes more ordered and adopts a closed conformation. When the product is created and ready to be released, the loop becomes more flexible and adopts the open conformation (Ramasubbu et al., 1996).
Domain B is found from residues 100 to 168 and exists as a protruding loop that extends from domain A. It is a mixture of helical and extended structures, and contains the calcium ion that is essential for the salivary alpha-amylase to function. The calcium ion binds to Asn-100, Arg-158, Asp-167, and His-201. The calcium ion makes the B domain partially rigid, which is enhanced by two disulfide bridges and an interdomain disulfide bond. This anchors the B domain near the substrate-binding cleft, which provides an asymmetric environment in the cleft so that the substrate binds in the proper orientation of the reducing and non-reducing ends(Ramasubbu et al., 1996).
Domain C is the C-terminal domain and is made up of 92 amino acids. It is made up of ten beta strands, eight of which are in the Greek key barrel formation and two of which are in loops. This domain is furthest from the active site. At the interface of the A and C domains there is a region high in hydrophobic molecules and groups of aromatic-aromatic interactions that occur in this area interspersed with methionine residues. Domain C contains Ser-414, a site for possible N-glycosylation. The salivary alpha-amylase exists in a glycosylated state, but human pancreatic amylase does not. The same gene does not produce both amylases, and in addition, it is possible that the occurrence of glycosylated proteins results from competition between protein folding and glycosylation events (Ramasubbu et al., 1996).