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Human α-amylase and starch digestion: An interesting marriage

Human α-amylase and starch digestion: An interesting marriage

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R
EVIEW
Human
a
-amylase and starch digestion: An interestingmarriage
Peter J. Butterworth, Frederick J. Warren and Peter R. Ellis
King’s College London, School of Medicine, Diabetes and Nutritional Sciences Division, Biopolymers Group, London, UK
a
-Amylasecatalyses thefirststep inthe digestionof starch,amain sourceofcarbohydrateinthe human diet. Amylase present in human saliva was one of the first enzymes ever to berecognisedbutmanypuzzlesremainaboutthemolecular mechanismsinvolvedinamylolysisof starch and even of the physiological role of the salivary amylase itself. Native starchgranules represent a formidable challenge for attack from an enzyme in solution. Moreoverthe frequently reported differences in the susceptibility to amylolysisof starches from variousbotanical species, plus the changes that occur in starch structure and properties duringdomestic and commercial food processing means that studies of the enzymology of starchdigestion can be challenging. We review the molecular properties of mammalian
a
-amylaseincluding its genetics, and speculate on the role of salivary amylase in digestion of dietarystarch.Alsoconsideredareenzymekineticapproachesthathavebeenusedinvitrostudiesofamylase digestion of starches. Such studies can result in better understanding of reasons forthe differences in glycaemic responses of humans following ingestion of different starchcontaining foods.
Received: December 6, 2010Accepted: January 5, 2011
Keywords:
a
-Amylase / Genetics / Kinetics / Salivary / Starch digestion
1 Introduction
In 1831, Erhard Leuchs reported that starch is brokendown when mixed with human saliva and used the namedptyalin to describe the agent in saliva that was responsiblefor the chemical reaction. This seems to have been one ofthe earliest accounts of an enzyme experiment and soonafter, in 1833, Payen and Persoz isolated an enzyme frombarleythatbrokedownstarch and namedit diastase.Thusenzymes now named amylases have a long history andmost readers will have memories from school biologyclasses of spitting into test tubes containing starchsuspensions stained dark blue with iodine and observingthedisappearanceofthecolourasthestarchishydrolysedin the presence of salivary amylase.Despite this long history and apparent familiarity withamylase properties, there are still many aspects of amy-laseandstarchbiochemistrythatcontinuetopuzzleinves-tigators including the full physiological significance of thatfamiliar amylase in saliva. Starch is normally the mainsource of digestible carbohydrate in the human diet andas such, is the major source of glucose that appearsat relatively high concentrations in the blood circulationfollowing intestinal digestion of a starch-containing meal.The first stage in the metabolism of starch is catalysed by
a
-amylase which progressively brings about hydrolysis ofthe polysaccharide resulting in the production of maltose,maltotriose and limit dextrins as the main products [1].Considerable differences, however, can occur in the post-prandial blood glucose and the corresponding insulinresponse, to the ingestion of different foods containingidentical amounts of starch [2]. That such differences
Correspondence:
Dr. Peter J. Butterworth, King’s CollegeLondon, School of Medicine, Diabetes and Nutritional SciencesDivision, Biopolymers Group, Franklin Wilkins Building,150 Stamford Street, London SE1 9NH.
E-mail:
peter.butterworth@kcl.ac.uk
Fax:
þ
44-207-848-4170
Abbreviations: AM,
amylose;
AP,
amylopectin;
CE,
catalyticefficiency;
RDS,
rapidly digested starch;
RS,
resistant starch.
DOI 10.1002/star.201000150
Starch/Sta¨  rke
2011,
63
, 395405 395
ß
2011WILEY-VCHVerlagGmbH&Co.KGaA,Weinheim www.starch-journal.com
 
occur is evidence of large variations in the rate and extentof starch digestion in the gastrointestinal tract [3–6].Attenuating the fluctuations in postprandial glycaemiaand insulinaemia is important in the prevention and treat-ment of life-style associated diseases, notably diabetesmellitus and cardiovascular disease, and also has implica-tions for obesity management [7–10].The search for full explanations for the differences inthe rates of digestion of starch-containing foods continuesto be a main focus for research, but inter-alia, the differ-encesaremainlyattributedtoinherentdissimilaritiesinthestructure of starch granules of different botanical speciesandfromstructuralalterationssubsequenttodomesticandcommercial processing of foods [11].Native starch is packaged in granules that are semi-crystallineandwhichareverylargecomparedwiththesizeof the
a
-amylase molecule. This contrasts with the vastmajority of intracellular metabolic enzymes where theenzyme molecules are usually huge in size compared withthemetabolitesthattheyactupon.Thestarchgranulesizeseemingly presents a very favourable target for attack byamylase with many potential sites for binding of theenzyme. In spite of this apparent binding advantage, thecomplete breakdown of starch within an intact granule is afairly slow process. Crystalline areas tend to be unfavour-able for enzyme attack and, in addition, the granules maycontain small but variable amounts of proteins and lipidsthat can also hinder starch–amylase interaction. Most ofthe starch consumed by humans will have been cookedand/or subjected to various processes during food pro-duction that disrupts the granules to a greater or lesserextent, but raw starch is consumed in many animal feeds.Processing that disrupts general granule integrity andreduces the degree of crystallinity, increases the suscepti-bility to amylase.In recent years, considerable advances have beenmade in knowledge of the structures of starch and mam-malian
a
-amylasesandmuchhasalsobeenlearnedaboutthe genes coding for
a
-amylase and their tissue expres-sion. It seems timely, therefore, to review relevant aspectsof what is known about starch and the enzyme to seewhether certain uncertainties in the starch-amylase storycan now begin to be clarified and also perhaps point upaspects of this subject that still need to be resolved byfurther experimentation.
2 Human
a
-amylase genes
The genes for
a
-amylase have been cloned by severalgroups and the article by Meiser and Ting [12] provides anexcellent review of the subject. Salivary glands and thepancreas express the enzyme in abundance but amylaseexpressioncanalsobefoundinthejejunumandmammarygland[13]. Inhumans andother mammals,twodistinctbutclosely related genes code for amylase. AMY1, the sali-vary gene, produces the enzyme present in saliva andmammaryglandandAMY2producestheenzymesynthes-ised in the pancreas that is secreted into the duodenum indigestive juices [13]. The expression of AMY1 can alsooccur in certain tumour tissues, e.g. lung. In humans bothAMY1 and AMY2 are located on the short arm ofchromosome 1 [14–16]. The coding regions of AMY1and AMY2 are very similar but the 5
0
untranslated regionof AMY2 is shorter than the equivalent region in AMY1[12]. The genes code for enzymes with MWs of about56 000 and the amino acid sequences of the salivaryand pancreatic enzymes differ by only 3%. Both AMY1and AMY2 are polymorphic and alloenzyme forms ofthe salivary and pancreatic amylases have been detected[12, 13]. The two pancreatic forms are exclusive to thepancreas and are expressed at nearly equal levels inthe gland [12]. The secreted salivary amylase has the
-terminal blocked by a pyroglutamic acid residue [17]that may confer some resistance to proteolysis.The organisation of the amylase gene cluster shownbelow is based on published data [12]. The genes arearranged in a head to tail orientation except for AMY1Bwhich is arranged in the reverse order. AMY1P is a pseu-dogene that lacks the first three exons of AMY1 [13].
AMY2____AMY2A____AMY1A____AMY1B____AMYP1____AMY1C
The promoter regions upstream of all the amylase genescontain a
g
-actin pseudogene and this finding has beeninterpreted to indicate that all the amylase genes arederived from a single ancestral gene [18]. In all exceptAMY2B, the
g
-actin pseudogene is interrupted by a retro-viral-like sequence and this element probably serves aregulatory role in the expression of the salivary enzymes[12]. Meisler and Ting [12] suggest that an ancestral mam-malian pancreatic gene gave rise to an ancestral AMY2gene which in turn, after
g
-actin and retroviral insertion,gave rise to the present AMY1 and AMY2 forms.A remarkable feature is that the copy number of theamylasegenesisvariable[12,19].For theAMY1(salivary)gene, variations in copy number from 2 to 15 have beendetected from the screening of 50 individuals [20].
3
a
-Amylase structures and properties
X-ray crystallography has been used to obtain 3D struc-tures of the
a
-amylase enzymes from human pancreas[21] and saliva [17] and from porcine pancreas [22]. Thestructures of these enzymes are all very closely related.Mammalian amylases are composed of three structuraldomains, the largest of which (domain A) contains import-ant active site residues that include two aspartate and one396 P. J. Butterworth
et al. Starch/Sta¨  rke
2011,
63
, 395–405
ß
2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.starch-journal.com
 
glutamate residue. Domain A also contains a bound Cl
À
ion. The activation of amylase by chloride has long beenknown [23, 24]. Domain B borders the active site regionandcontainsaboundCa
2
þ
ionthatisprobablyimportantinmaintainingproteinconformationintheregionoftheactivesite. Domain C at the
-terminal region of the moleculeappears unlikely to play a direct role in the catalytic mech-anism. It is less firmly associated with domains A and B[21, 25].The catalytic event in amylases is an example of adouble displacement mechanism and carboxyl groups ofaspartate and glutamate residues function as acid/basecatalystsandasanucleophilicreactantfor theformationofacovalentintermediateduringthecatalyticsequence.Theactivation by a Cl
À
ion may come about by facilitating theprotonation of a catalytically important carboxyl groupthrough a charge relay-system [26–28].
a
-Amylase is an endoenzyme that carries out multipleattack on linear portions of AM and AP with maltose andmaltotriose as the principal short chain products [1, 29].Only minor amounts of glucose are produced [1, 30].Kinetic studies suggested that the active site of porcinepancreatic enzyme can accommodate up to five glucoseresidues, i.e. there are five sub-sites at which glucoseresidues can become bound [1]. The existence of multiplesites was confirmed by the production of 3D structuresfrom X-ray crystallography, although a report of the struc-ture of pig pancreatic amylase complexed with the potentcarbohydrate inhibitor acarbose provided evidence of asixth site [22]. The glucosidic bond susceptible to attackis that linking residues 3 and 4 (Fig. 1) and the catalyticallyimportant aspartate and glutamate residues of theactivesitearesuitablylocatedforattackonthesusceptiblelink.Kinetic studies using malto-oligosccharides as sub-strates[31]wereusedtodeterminethebindingfreeenergy(
D
G
) associated with each of the five sites.
D
G
valuesbecome more negative (from approx
À
5 to
À
16 kJ/mol),i.e.bindingbecomesstronger,asthesub-sites1–5becomefilled with glucose residues in a substrate [30–32]. Thebinding energy of site 3 is
þ
17.6 kJ/mol and this unfavour-able condition probably arises from forced distortion of theglucan ring on binding. The binding energies are similar tothose observed for the sites on lysozyme [33]. The totalfree energy of binding accruing from occupancy of all thesubsites is of consequence when considering the action ofamylase on native and hydrothermally treated starch (seebelow).
4 Starch structure and implications for
a
-amylase action
Descriptions and discussion of starch structure and prop-erties are covered only briefly in this review. The infor-mation presented here is that which seems most pertinentto understanding the role of amylase in starch digestion.The literaturecontainsmanyexcellent generalaccounts ofstarch structure both in its native state and of the changesthat occur in structure resulting from various methods ofprocessing and readers are referred to these for moredetailed information [34–41].Native starch granules are semi-crystalline with amor-phous regions largely composed of AM and the branchpointsinAPmolecules,andcrystallitesformedfromlongerchains of AP that twist into H-bonded double helices.Crystalline regions are of two polymorphic types, A andB [42–45], that differ in the packing of the helices. Thehelices in A are orthogonally arranged whereas there ishexagonal packing in B starches [43, 44]. The number ofassociatedwater moleculesisgreaterintheBcrystals[43,44]. Cereal starches have A type crystallinity and tuberstarches such as potato possess the B polymorph,whereas legume starches have a so-called C pattern ofcrystallinitythatisknowntoarisefromthepresenceofbothAandBpolymorphsindifferentregionsofthegranule[39].AM is enriched towards the periphery of the granules andthis peripheral material has shorter chain lengths than AMlocated more centrally within the granule [46]. Granulesfrom cereal starches, e.g. maize, wheat and barley, havesurface pores that open onto channelswith diameters thatrange in size from 5 to 400 nm [46].Native starches with a high B polymorph content, e.g.potato,areattackedslowlybyamylasebutcanbedigestedmore rapidly after hydrothermal processing [47]. Native Atype starches tend to be more favourable substrates [47].During domestic and commercial food processing, gran-ules can be subjected to varying degrees of hydrothermaltreatments which cause granules to swell by absorption ofwater and crystalline structures are disrupted with anincrease in amorphous material [41]. The increase ofamorphous material makes the starch more digestibleby
a
-amylase [11]. Continued heating in aqueous
Figure 1.
Subsites at the active centre of
a
-amylase.Bond breakage occurs between residues 3 and 4.Carboxyl groups of key aspartate and glutamate residuesfunction as acid-base and nucleophilic catalysts [22].
Starch/Sta¨  rke
2011,
63
, 395405 397
ß
2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.starch-journal.com

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