Amylase
IntroductionIntroduction
Discovered and isolated by Anselme Payen in 1833, amylase was the first enzyme to be discovered[1]. Amylases are hydrolases, acting on α-1,4-glycosidic bonds[2]. They can be further subdivided into α,β and γ amylases[1]. α-Amylase is an enzyme that acts as a catalyst for the hydrolysis of alpha-linked polysaccharides into α-anomeric products[3]. The enzyme can be derived from a variety of sources, each with different characteristics. α-Amylase found within the human body serves as the enzyme active in pancreatic juice and salvia[2]. α-Amylase is not only essential in human physiology but has a number of important biotechnological functions in various processing industries.
Structure[3]Structure[3]
Shown as 1hvx is the structure of the thermostable α-amylase of Bacillus stearothermophilus (BSTA)[3]. BSTA is comprised of a single polypeptide chain. This chain is folded into three domains: A, B and C. These domains are generally found on all α-amylase enzymes. The constitutes the core structure, with a (β/α)8-barrel.The consists of a sheet of four anti-parallel β-strands with a pair of anti-parallel β-strands. Long loops are observed between the β-strands. Located within the B domain is the for Ca2+-Na+-Ca2+. consisting of eight β-strands is assembled into a globular unit forming a Greek key motif. It also holds the Ca2+ binding site in association with domain A. Positioned on the C-terminal side of the β-strands of the (β/α)8-barrel in domain A is the active site. The catalytic residues involved for the BSTA active site are . The residues are identical to other α-amylases, yet there are positional differences which reflect the flexible nature of catalytic resides. found in the interior of domain B and at the interface of domain A and C, constitute the metal ion binding sites. All α-amylases contain one strongly conserved Ca2+ ion for structural integrity and enzymatic activity.[4] CaI is consistent in α-amylases, however there are structural differences between the linear trio of CaI, CaII and Na in other enzymes. CaIII acts as a bridge between two loops, one from α6 of domain A, and one between β1 and β2 of domain C.
Chloride Dependent EnzymesChloride Dependent Enzymes
A family of chloride-dependent enzymes, including salivary and pancreatic α-amylase, require the binding of a chloride ion to be allosterically activated[4]. The function of the chloride ion still remains uncertain. No relationship has been observed between the anion binding affinity and its activity, indicating the complexity between the binding parameters and mechanism it activates[4]. Studies have shown that nitrite and nitrate ions with pancreatic α-amylase fit within the chloride binding site, thus making all the necessary hydrogen bonds and enhancing the relative activity by 5-fold[5].
FunctionFunction
MechanismMechanism
In the human body, α-amylase is part of digestion with the breakdown of carbohydrates in the diet. The mechanism involved includes catalyzing substrate hydrolysis by a double replacement mechanism, forming a covalent glycosyl-enzyme intermediate and hydrolyzed through oxocarbenium ion-like transition states[6]. One of the carboxylic acids in the active site acts as the catalytic nucleophile during the formation of the intermediate. A second carboxylic acid operates as the acid/base catalyst, supporting the stabilization of the transition states during the hydrolysis[6].
Human Salivary and Pancreatic α-AmylaseHuman Salivary and Pancreatic α-Amylase
Salivary α-Amylase hydrolyzes the (α1-4) glycosidic linkages of starch, separating it into short polysaccharide fragments[7]. Once the enzyme reaches the stomach, it becomes inactivated due to the acidic pH. Further breakdown of starch occurs by secretion of a second form of the enzyme by the pancreas. Pancreatic juice enters the duodenum and pancreatic α-amylase further cleaves starch to yield maltose, maltotriose and oligosaccharides[7]. The oligosaccharides are referred to as dextrins, which are fragments of amylopectin consisting of (α1-6)branch points[7]. Microvilli of the intestinal epithelia break maltose and dextrins into glucose, which gets absorbed into the circulatory system[7]. Glycogen has a relatively similar structure as starch, and thus proceeds in the same digestive pathway.
RegulationRegulation
α-Amylase is regulated through a number of inhibitors. These inhibitors are classified according to six categories, based on their tertiary structures[8]. Inhibitors of α-amylase block the active site of the enzyme. In animals, inhibitors control the conversion of starch to simple sugars during glucose peaks after a meal so that breakdown of glucose occurs at a rate the body can handle[8]. This is particularly important for diabetics, who require low quantities of α-amylase to maintain control over glucose levels. After taking insulin however, pancreatic α-amylase escalates. Plants use these inhibitors as a defense mechanism to inhibit the use of α-amylase in insects, thus protecting themselves from herbivory[9].
Industrial UsesIndustrial Uses
α-Amylase is used extensively in various industrial processes. In textile weaving, starch is added for warping. After weaving, the starch is removed by Bacillus subtilis α-amylase[1]. Dextrin, which is a viscosity improver, filler, or ingredient of food, is manufactured by the liquefaction of starch by bacteria α-amylase[1]. Bacterial α-amylases of B.subtilis, or B.licheniformis are used for the initial starch liquefaction in producing high conversion glucose syrup[1]. Pancreatitis can be tested by determining the level of amylases in the blood, a result of damaged amylase-producing cells, or excretion due to renal failure[10]. α-Amylase is used for the production of malt, as the enzyme is produced during the germination of cereal grains[1].
3D structures of Alpha-amylase3D structures of Alpha-amylase
Alpha-amylaseAlpha-amylase
3ij7, 1xv8, 1c8q, 1bsi, 1smd, 1hny – hAAM – human
1xgz, 1q4n, 1kb3, 1kbb, 1kbk, 1kgu, 1kgw, 1kgx, 1jxj, 1jxk, 2cpu - hAAM (mutant)
3n8t – TkAAM – Thermococcus kodakarensis
3k8k – BtAAM – Bacterioides thetaiotaomicron
3kwx, 2guy – AoAAM – Aspergilluys oryzae
2wpg – AAM – Xanthomonas campestris
2wc7, 2wcs, 2wkg – AAM catalytic region – Cyanobacterium
3bh4 – BaAAM – Bacillus amyloliquefaciens
3bsg – bAAM (mutant)
3dhu – AAM – Lactobacillus plantarum
3dc0 – AAM – Bacillus KR8104
3bcf, 1wza – HoAAM – Halothermothrix orenii
2die, 1wp6 – AAM alkaline – Bacillus sp.
1ud2, 1ud4, 1ud5, 1ud6, 1ud8 - AAM – Bacillus sp. KSM-K38
1ud3 – AAM (mutant) – Bacillus sp. KSM-K38
2gjr – BhAAM – Bacillus halmapalus
2b5d – AAM – Thermotoga maritima
2c3g, 2c3v – BhaloAAM – Bacillus halodurans
1ji1, 1ji2, 1bvz - TvAAM – Thermoactinomyces vulgaris
1wzk, 1wzl, 1wzm, 1izj, 1izk, 1jf5, 1jf6 – TvAAM (mutant)
1mwo, 1mxd – PwAAM – Pyrococcus woesei
1ob0, 1bli - BlAAM (mutant) – Bacillus licheniformis 1vjs – BlAAM precursor
1b0i, 1aqm, 1aqh – PhAAM - Pseudoalteromonas haloplanktis
1g5a – NpAAM – Neisseria polysaccharea
1hvx - BaAAM – Bacillus stearothermophilus
1qho – GsAAM – Geobacillus stearothermophilus
1jae – TmAAM – Tenebrio molitor
1pif – pAAM – pig
AAM binary complexes
1xd0, 1xd1, 1cpu, 1jfh - hAAM + saccharide
1b2y, 1xcw, 1xcx - hAAM + acarbose
3blk, 3blp, 1z32, 1nm9, 1mfu, 1mfv, 3cpu - hAAM (mutant) + saccharide
3dhp, 1xh0, 1xh2 - hAAM (mutant) + acarbose
3ij8, 3ij9 – hAAM catalytic intermediate
3baw – hAAM + N3
3bax - hAAM (mutant) + N3
3bak – hAAM (mutant) + NO3
1xh1 - hAAM (mutant) + Cl
2qv4, 3baj, 3bay - hAAM + acarbose + NO2
3old, 3ole, 3olg, 3oli – hAAM + statin
1u2y, 1u30, 1u33 – hAAM + inhibitor
3n92, 3n98 – TkAAM + saccharide
3l2l, 3l2m, 1vah, 1wo2, 1ua3, 1pig, 1ppi - pAAM + saccharide
1hx0 – pAAM + acarbose
1kxq, 1kxt, 1kxv – pAAM + antibody VHH fragment
1bvn, 1dhk – pAAM + protein inhibitor
1ose – pAAM + acarbose
3k8l - BtAAM (mutant) + saccharide
3k8m - BtAAM + acarbose
2d2o, 1jl8, 1jib - TvAAM + saccharide
3a6o – TvAAM + acarbose
2d0f, 2d0g, 2d0h, 1vb9, 1vfm, 1vfo, 1vfu, 1uh2, 1uh4 - TvAAM (mutant) + saccharide
1uh3 - TvAAM (mutant) + acarbose
1ava – bAAM + protein inhibitor
1bg9, 1rpk - bAAM + acarbose
1p6w – bAAM + substrate analog
1rp8, 1b1y, 1rp9 - bAAM (mutant) + saccharide
3bsh, 2qpu, 2qps - bAAM (mutant) + acarbose
3bcd - HoAAM + saccharide
3bc9 - HoAAM + acarbose
2gjp, 1w9x - BhAAM + saccharide
2gvy - AoAAM + saccharide
1zs2, 1s46, 1mvy, 1mw0, 1mw1, 1mw2, 1mw3 - NpAAM (mutant) + saccharide
1bag - BsAAM + saccharide – Bacillus subtilis
1ua7 – BsAAM + acarbose
2d3l, 2d3n - BacAAM + saccharide – Bacillus
2c3h, 2c3w, 2c3x - BhaloAAM + saccharide
1mxg - PwAAM + acarbose
1g9h, 1g94 - PhAAM + saccharide
1kxh - PhAAM (mutant) + acarbose
1l0p – PhAAM + NO3
1e40 – BaAAM chimera + saccharide
1e3z - BaAAM chimera + acarbose
1fa2 - AAM + saccharide – Sweet potato
1qhp - GsAAM + saccharide
1clv, 1viw, 1tmq – TmAAM + protein inhibitor
1gah, 1gai – AaAAM + acarbose – Aspergillus awamori
3gly, 1agm, 1glm – AaAAM + saccharide
Pullulanase alpha-amylasePullulanase alpha-amylase
2wan – AAM – Bacillus acidopullululyticus
2fgz – KaAAM – Klebsiella aerogenes
2e8y - BsAAM
Pullulanase alpha-amylase binary complexes
2fh6, 2fh8, 2fhb, 2fhc, 2fhf - KaAAM + saccharide
3fax - AAM + saccharide – Streptococcus agalactiae
2e8z, 2e9b - BsAAM + saccharide
1g1y - TvAAM (mutant) + saccharide
Beta-amylaseBeta-amylase
2xfr – bBAM
1wdp – sBAM – soybean
2dqx, 1uko, 1ukp – sBAM (mutant)
1vem, 5bca, 1cqy, 1b90 – BcBAM – Bacillus cereus
1ven - BcBAM (mutant)
Beta-amylase binary complexes
2xff – bBAM + acarbose
2xfy, 2xg9, 2xgb, 2xgi – bBAM + inhibitor
1wdq, 1wdr, 1wds, 1v3h, 1v3i, 1q6d, 1q6e, 1q6f, 1q6g - sBAM (mutant) + saccharide
1q6c, 1bfn, 1bya, 1byb, 1byc, 1byd, 1btc - sBAM + saccharide
1j0y, 1j0z, 1j10, 1j11, 1j12, 1j18, 1b9z - BcBAM + saccharide
1veo, 1vep, 1itc - BcBAM (mutant) + saccharide
Gamma-amylaseGamma-amylase
1lf6 – TtGAM – Thermoanaerobacterium thermosaccharolyticum
1lf9 - TtGAM + acarbose
Maltohexaose-producing amylaseMaltohexaose-producing amylase
1wp6 - BacMAM
1wpc – BacMAM + saccharide
Maltogenic amylaseMaltogenic amylase
1gvi, 1sma – MAM – Thermus sp.
Taka amylaseTaka amylase
2taa – AoTAM
7taa – AoTAM + acarbose
ReferencesReferences
- ↑ 1.0 1.1 1.2 1.3 1.4 1.5 Yamamoto T.1988. Handbook of Amylases and Related Enzymes: Their Sources, Isolation Methods, Properties and Applications. Osaka Japan: Pergamon Press
- ↑ 2.0 2.1 Aghajari N, Feller G, Gerday C, Haser R. Crystal structures of the psychrophilic alpha-amylase from Alteromonas haloplanctis in its native form and complexed with an inhibitor. Protein Sci. 1998 Mar;7(3):564-72. PMID:9541387
- ↑ 3.0 3.1 3.2 Suvd D, Fujimoto Z, Takase K, Matsumura M, Mizuno H. Crystal structure of Bacillus stearothermophilus alpha-amylase: possible factors determining the thermostability. J Biochem. 2001 Mar;129(3):461-8. PMID:11226887
- ↑ 4.0 4.1 4.2 Aghajari N, Feller G, Gerday C, Haser R. Structural basis of alpha-amylase activation by chloride. Protein Sci. 2002 Jun;11(6):1435-41. PMID:12021442
- ↑ Maurus R, Begum A, Williams LK, Fredriksen JR, Zhang R, Withers SG, Brayer GD. Alternative catalytic anions differentially modulate human alpha-amylase activity and specificity(,). Biochemistry. 2008 Mar 18;47(11):3332-44. Epub 2008 Feb 20. PMID:18284212 doi:10.1021/bi701652t
- ↑ 6.0 6.1 Maurus R, Begum A, Williams LK, Fredriksen JR, Zhang R, Withers SG, Brayer GD. Alternative catalytic anions differentially modulate human alpha-amylase activity and specificity(,). Biochemistry. 2008 Mar 18;47(11):3332-44. Epub 2008 Feb 20. PMID:18284212 doi:10.1021/bi701652t
- ↑ 7.0 7.1 7.2 7.3 Kuriki T, Imanaka T. The concept of the alpha-amylase family: structural similarity and common catalytic mechanism. J Biosci Bioeng. 1999;87(5):557-65. PMID:16232518
- ↑ 8.0 8.1 PPMID: 17713601
- ↑ Franco OL, Rigden DJ, Melo FR, Grossi-De-Sa MF. Plant alpha-amylase inhibitors and their interaction with insect alpha-amylases. Eur J Biochem. 2002 Jan;269(2):397-412. PMID:11856298
- ↑ Yang RW, Shao ZX, Chen YY, Yin Z, Wang WJ. Lipase and pancreatic amylase activities in diagnosis of acute pancreatitis in patients with hyperamylasemia. Hepatobiliary Pancreat Dis Int. 2005 Nov;4(4):600-3. PMID:16286272