Starch in food
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ß 2004, Woodhead Publishing Limited
Starch in food
Structure, function and applications
Edited by
Ann-Charlotte Eliasson
ß 2004, Woodhead Publishing Limited
Published by Woodhead Publishing Limited
Abington Hall, Abington
Cambridge CB1 6AH
England
www.woodhead-publishing.com
Published in North America by CRC Press LLC
2000 Corporate Blvd, NW
Boca Raton FL 33431
USA
First published 2004, Woodhead Publishing Limited and CRC Press LLC
ß 2004, Woodhead Publishing Limited
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Contributorcontactdetails
PartIAnalysingandmodifyingstarch
1Plantstarchsynthesis
J. Preiss, Michigan State University, USA
1.1Introduction:localizationandfunctionofstarchinplants
1.2 Starch synthesis: enzyme reactions in plants and algae
andglycogensynthesisincyanobacteria

1.3Propertiesofplantglucansynthesizingenzymes:
ADP-glucosepyrophosphorylase
1.4Propertiesofplantglucansynthesizingenzymes:
starchsynthase
1.5Propertiesofplantglucansynthesizingenzymes:
branchingenzymes
1.6Initiationofstarchsynthesisusingaglucosyl-protein
1.7Locatingstarchsynthesisinplants:theplastid
1.8Invivosynthesisofamylopectin
1.9Regulatingstarchsynthesisinplants
1.10References
2Analysingstarchstructure
E. Bertoft, A
Ê
bo Akademi University, Finland
2.1Introduction:characterisingstructuresofstarchcomponents
2.2Fractionationofstarch
2.3Analysisofamylose
Contents
ß 2004, Woodhead Publishing Limited
2.4Analysisofamylopectinstructure
2.5Analysisofintermediatematerials
2.6Analysisofchemicallymodifiedstarches
2.7Futuretrends
2.8Sourcesoffurtherinformationandadvice
2.9References
3Starchbioengineering
A. Blennow, The Royal Agricultural and Veterinary University,
Denmark
3.1Introduction:theimportanceofstarch

3.2Technologiesforgeneticmodificationandstarchprofiling
3.3Improvingstarchyieldandstructure
3.4Physicalandchemicalpropertiesofmodifiedstarches
3.5Functionalityandusesofmodifiedstarchesin
foodprocessing
3.6Ensuringsuccessfulmodificationofstarch
3.7Futuretrends
3.8References
4Starch-actingenzymes
D. P. Butler, Marc J. E C. van der Maarel and P. A. M. Steeneken,
TNO Nutrition and Food Research Institute, The Netherlands
4.1Introduction:theimportanceofenzymes
4.2Usingenzymestomodifystarch
4.3 Developing starch-modifying enzymes for food
processingapplications
4.4Futuretrends
4.5References
5Understandingstarchstructureandfunctionality
A. M. Donald, University of Cambridge, UK
5.1Introduction:overviewofpackingatdifferentlengthscales
5.2Theeffectofamylopectinchainarchitectureonpacking
5.3Improvingpackingwithinstarchgranules
5.4Thegelatinisationprocess
5.5Foodprocessing:implicationsofstarchgranulestructure
5.6Conclusionsandfuturetrends
5.7Sourcesoffurtherinformationandadvice
5.8References
6Measuringstarchinfood
M Peris-Tortajada, Polytechnic University of Va lencia, Spain
6.1Introduction

6.2Samplepreparation
ß 2004, Woodhead Publishing Limited
6.3Methodsofanalysingstarchinfood
6.4 Determining starch in food: recent technological
developments
6.5Futuretrends
6.6Sourcesoffurtherinformationandadvice
6.7References
PartIISourcesofstarch
7Thefunctionalityofwheatstarch
H. Cornell, RMIT University, Australia
7.1 Introduction: manufacture of wheat starch for the
foodindustry
7.2Granularandmolecularstructureofwheatstarch
7.3Functionalityofwheatstarch:granules,filmsandpastes
7.4Rheologicalpropertiesofstarchpastesandgels
7.5 Improving and chemically modifying wheat starch for use
inthefoodindustry
7.6Wheatstarchsyrups
7.7Analysingstarch-basedproducts
7.8Futuretrends
7.9Sourcesoffurtherinformationandadvice
7.10References
8Developmentsinpotatostarches
W. Bergthaller, Federal Centre for Nutrition and Food, Germany
8.1Introduction
8.2Componentsandrheologicalpropertiesofpotatostarch
8.3Techniquesforproducingpotatostarch
8.4 Improving the functionality of potato starch for use in
thefoodindustry

8.5Futuretrends
8.6References
9Thefunctionalityofricestarch
J. Bao and C. J. Bergman, Texas A&M University, USA
9.1Introduction
9.2Riceflourandstarchasfoodingredient
9.3Constituentsofricestarch
9.4Structureandfunctionalityofricestarch
9.5Gelatinizationandthestructureofricestarch
9.6Retrogradationandotherpropertiesofricestarch
9.7 Improving rice starch functionality for food
processingapplications
9.8Futuretrends
ß 2004, Woodhead Publishing Limited
9.9Sourcesoffurtherinformationandadvice
9.10References
10Newcornstarches
P. J. White and A. Tzioti s, Iowa State University, USA
10.1Introduction:theuseofcornstarchinfoodprocessing
10.2 Improving the functionality of corn starch for food
processingapplications:naturalcornendospermmutants
10.3 Chemically modifying corn starches for use in the
foodindustry
10.4 Genetically modifying corn starches for use in the
foodindustry
10.5Futuretrends
10.6Sourcesoffurtherinformationandadvice
10.7References
11Tropicalsourcesofstarch
S.N.Moorthy,CentralTuberCropsResearchInstitute,India

11.1Introduction:tropicalsourcesofstarch
11.2Characteristicsandpropertiesofcassavastarch
11.3Characteristicsandpropertiesofsweetpotatostarch
11.4Characteristicsandpropertiesofyamandaroidstarches
11.5Characteristicsandpropertiesofotherminorrootstarches
11.6Modifying`tropical'starchesforuseinthefoodindustry
11.7Futuretrends
11.8References
PartIIIApplications
12Starchasaningredient:manufactureandapplications
P. Taggart, National Starch and Chemical, UK
12.1Introduction
12.2Manufacture
12.3Structure
12.4Modifications
12.5Technicaldata
12.6Usesandapplications
12.7Regulatorystatus:Europeanlabeldeclarations
12.8Acknowledgements
12.9Bibliography
13Utilizingstarchesinproductdevelopment
T. Luallen, Cargill Inc., USA
13.1Introduction
ß 2004, Woodhead Publishing Limited
13.2Componentsofstarch
13.3Foodapplicationsfornaturalandmodifiedstarches
13.4Methodsofstarchselection
13.5Factorsaffectingstarchinfoodproducts
13.6 Using the functional properties of starch to enhance
foodproducts

13.7References
14Modifiedstarchesandthestabilityoffrozenfoods
H. D. Goff, University of Guelph, Canada
14.1Introduction
14.2Thestructureandstabilityoffrozenfoods
14.3Theroleofmodifiedstarchinstabilizingfrozenfoods
14.4Futuretrends
14.5Sourcesoffurtherinformationandadvice
14.6References
15Starch-lipidinteractionsandtheirrelevanceinfoodproducts
A-C. Eliasson and M. Wahl gren, Lund University, Sweden
15.1Introduction
15.2Thestructureandpropertiesofthestarch-lipidcomplex
15.3Analysisofstarch:lipidsandemulsifiers
15.4Theeffectoflipidsonstarchbehaviour
15.5Enzymaticdegradationofamylose-lipidcomplexes
15.6Futuretrends
15.7References
16Starch-basedmicroencapsulation
P. Forssell, VTT Biotechnology, Finland
16.1Introduction:usingmicroencapsulationinfoodprocessing
16.2 Using starch in microencapsulation: starch hydrolysates,
derivatives,polymersandgranules
16.3Starch-basedshellmatricesforfoodingredients
16.4Futuretrends
16.5References
PartIVStarchandhealth
17 Development of a range of industrialised cereal-based
foodstuffshighinslowlydigestiblestarch
V. Lang, Danone Vitapole, France

17.1Introduction
17.2Characteristicsandpropertiesofstarchandstarchyfoods
17.3LowGIdietsandtheirassociatedhealthbenefits
ß 2004, Woodhead Publishing Limited
17.4 Case study: low glyc aemic index, high slowly digestible
starch plain biscuits, the EDP
Õ
(`Long-lasting energy')
rangedevelopedbyDanone,Vitapole
17.5Futuretrends
17.6Sourcesoffurtherinformationandadvice
17.7Acknowledgements
17.8References
18Starch:physicalandmentalperformance
F. Brouns, Cerestar Vilvoorde R & D Centre, Belgium and
University of Maastricht, Netherlands and L. Dye, University of Leeds,
England
18.1Introduction
18.2Physicalperformance:energyrequirements,delivery
andavailability
18.3Mentalperformance:theeffectsofglucose
18.4 Mental performance: the effects of CHO and glucose
duringtheday
18.5Futuretrends
18.6References
19Detectingnutritionalstarchfractions
K. Englyst and H. Englyst, Englyst Carbohydrates, UK
19.1Introduction
19.2MethodsofdeterminingRAG,SAGandRSfractions
19.3Qualitycontrolandtroubleshooting

19.4Carbohydratebioavailabilitydataforselectedfoods
19.5Conclusionandfuturetrends
19.6Acknowledgement
19.7References
20Resistantstarch
M. Champ, INRA-UFDNH/CRNH, France
20.1Introduction
20.2Effectsofresistantstarchonthedigestivesystem
20.3Improvingthefunctionaleffectsofresistantstarch
20.4Futuretrends
20.5Sourcesoffurtherinformationandadvice
20.6References
21Analysingstarchdigestion
R. E. Wachters-Hagedoorn, M. G. Priebe and R. J. Vonk,
University Hospital Groningen, The Netherlands
21.1Introduction
21.2Starchandthepreventionofhypo-andhyperglycemia
ß 2004, Woodhead Publishing Limited
21.3 The determinants of the rate of absorption of
starch-derivedglucose
21.4Techniquesformonitoringstarchdigestion
21.5 Current applications of slowly available starch and the
preventionofhyper-andhypoglycemia
21.6Futuretrends
21.7Sourcesoffurtherinformationandadvice
21.8References
ß 2004, Woodhead Publishing Limited
Chapter 1
Professor J. Preiss
Department of Biochemistry

Michigan State Univer sity
East Lansing
MI 48824
USA
Tel: 517 353 3137
Fax: 517 353 9334
E-mail:
Chapter 2
Dr E. Bertoft
Department of Biochemistry and
Pharmacy
A
Ê
bo Akademi University
PO Box 66
FIN 20521
Turku
Finland
Tel: 358 2 215 4272
Fax: 358 2 215 4745
E-mail:
Chapter 3
Dr A. Blennow
The Royal Agricultural and
Veterinary University
Denmark
E-mail:

Chapter 4
Dr D. P. Butler, Dr M. J. E. C. van der

Maarel and Dr P. A. M. Steeneken
TNO Nutrition and Food Research
Institute
Groningen
The Netherlands
E-mail:

Contributor contact details
ß 2004, Woodhead Publishing Limited
Chapter 5
Professor A. Donald
Department of Physics
Cavendish Laboratory
University of Cambrid ge
Madingley Road
Cambridge
CB3 0HE
Tel: +44 1223 337382
Fax: +44 1223 337000
E-mail:
Chapter 6
Professor M. Peris-Tortajada
Department of Chemistry
Polytechnic University of Valencia
46071 Valencia
Spain
E-mail:
Chapter 7
Professor H. Cornell
Department of Applied Chemistry

RMIT University
City Campus
GPO Box 2476V
Melbourne
Victoria 3001
Australia
Tel: 0061 3-9925 2117
Fax: 0061 3-9039 1321
E-mail:
Chapter 8
Dr W. Bergthaller
Federal Research Centre for Nutrition
and Food
Location Detmoed and Muenster
Institute for Cereal, Potato and Starch
Technology
PO Box 1354
Detmold 32756
Germany
Tel: + 49 5231 741320
Fax: + 49 5231 741300
E-mail: ;

Chapter 9
Dr C. Bergman
University of Nevada
Las Vegas
Nevada 89154
USA
Dr J. Bao

Zhejiang University
Huajiachi
Hangzhou 310029
China
E-mail:

ß 2004, Woodhead Publishing Limited
Chapter 10
Professor P. J. White and Ms A.
Tziotis
Department of Food Science and
Human Nutrition and Centre for
Crops Utilization Research
2312 Food Sciences Building
Iowa State University
Ames
Iowa
USA
Tel: 515 294 9688
Fax: 515 294 8181
E-mail: pjwhite@iast ate.edu
Chapter 11
Dr S. N. Moorthy
Central Tuber Crops Research
Institute
Sreekariyam
Thiruvananthapuram
695 017 Kerala
India
Tel: 0471 598551

Fax: 0091 471 590063
E-mail:
Chapter 12
Dr P. Taggart
National Starch and Chemical Ltd
Prestbury Court
Green Court Business Park
333 Styal Road
Manchester
UK
Tel: +44(0)161 435 3200
Fax: +44(0)161 435 3221
E-mail:
Chapter 13
Dr T. Luallen
Cargill Inc
Specialty Food and Pharma Solutions
B U
PO Box 1467
Cedar Rapids
IA 52406
USA
Tel: 319 399 6187
Fax: 319 399 6123
E-mail:
Chapter 14
Professor H. D. Goff
Department of Food Science
University of Guelph
Guelph

Ontario N1G 2W1
Canada
Tel: 519 824 4120
Fax: 519 824 6631
E-mail:
Chapter 15
Professor A-C. Eliasson and
M. Wahlgren
Food Technology Division
Lund University
Box 124
22100 Lund
Sweden
Tel: +46 222 9674
Fax: +46 222 9517
E-mail: ann-

ß 2004, Woodhead Publishing Limited
Chapter 16
Dr P Forssell
VTT Biotechnology
Tietotie 2
PO Box 1500
02044 VTT
Finland
E-mail:
Chapter 17
Dr V. Lang
Danone Vitapole
R&D Center of the Groupe Danone

Nutrition Research Department
Route DeÂpartementale 128
91767 Palaiseau Cedex - France
Tel: +33 1 69 35 72 34
Fax: + 33 1 69 35 76 89
E-mail:
Chapter 18
Professor F. Brouns and Dr L. Dye
Cerestar Vilvoorde R&D Centre
Havenstraat 84
B-1800 Vilvoorde
Belgium
Tel: 00 32 2 2570736
Fax: 00 32 2 2570740
Email:
Chapter 19
Dr K. Englyst and Dr H. Englyst
Englyst Carbohydrates
2 Venture Road
Chilworth Science Park
Southampton
SO16 7NP
UK
Tel: +44 (0) 23 80 769650
Fax: +44 (0) 23 80 769654
Email:
Chapter 20
Dr M. Champ
INRA-UFDNH/CRNH
Rue de la GeÂraudieÁre

BP 71627
44316 Nantes Cedex 03
France
Email:
Chapter 21
Professor R. J. Vonk
Laboratory of Nutrition and
Metabolism
Laboratory Centre CMC V, Y2147
University Hospital Groningen
Hanzeplein 1
P.O. Box 30.001
9700 RB Groningen
The Netherlands
Tel: +31-50-3632675
Fax: +31-50-3611746
Email:
ß 2004, Woodhead Publishing Limited
Part I
Analysing and modifying starch
ß 2004, Woodhead Publishing Limited
1.1 Introduction: localization and function of starch in plants
This chapter reviews starch synthesis in higher plants and algae. Since the
reactions leading to glycogen synthesis in the cyanobacteria are similar to those
observed in the higher plants there will be some referral to studies in those
organisms particularly in regulation of cyanobacterial 1,4-glucan synthesis. The
enzymology and biochemistry of the various enzymes in the plant, algal and
cyanobacterial systems will also be described. In view of the existing information
available on the properties of the starch biosynthetic enzymes and the effects of
certain mutants on starch structure a pathway of starch synthesis is described which

postulates specific functions for the starch synthases and branching enzymes.
Finally regulation of starch synthesis at the enzymatic level will be discussed and
in relation to this regulation, recent results indicating how starch content has been
increased in certain plants will be descibed. A previous chapter
1
in the second
edition of Starch Chemistry and Technology which reviewed starch biosynthesis
discussed the various maize endosperm mutants or mutant combinations, 26 of
them, that showed an effect on the quantity or the nature of the starch formed. This
information remains of interest and the reader is referred to that review. However,
for many of those mutants the biochemical basis for mutation effects on starch
quantity or quality wa s unknown. This review will deal with only the mutants
where the biochemical process affected by the mutation has been elucidated to at
least some extent. There are some recent reviews on starch biosynthesis
2±11
that
discuss many of the areas presented in this chapter.
1.1.1 Leaf starch
Starch is deposited in granules in almost all green plants and in various types of
1
Plant starch synthesis
J. Preiss, Michigan State University, USA
plant tissues and organs, e.g., leaves, roots, shoots, fruits, grains, and stems.
ß 2004, Woodhead Publishing Limited
Illumination of the leaf in bright light causes the formation of starch granules in
the chloroplast organelle and was demonstrated in the nineteenth century.
12
Disappearance of the starch occurs either by exposure of the leaf to low light or
by extended exposure in the dark (24±48 hours). This is readily observed by
iodine staining of the tissue

13
or by light or electron microscopy.
14
Starch
accumulates due to carbon fixation during photosynthesis and the starch formed
in the light is degraded in the dark to products that are in most cases utilized for
sucrose synthesis. Mutants of Arabidopsis thaliana unable to synthesize starch,
grow at the same rate as the wild type in a continuous light regime because they
are able to synthesize sucrose,
15
but their growth rate is drastically reduced if
grown in a day-night regime. The reason for this is that the accumulated starch is
required for sucrose synt hesis at night; the sucrose is transported from the leaf to
the sink tissues. Biosynthesis and degradation of starch in the leaf is therefore a
dynamic process having diurnal fluctuations in its stored levels.
Starch also plays an important role in the operation of stomatal guard cells,
where it is degraded during the day. In the late afternoon or evening while the
stomata are open, the starch is resynthesized. Leaf starch is lower in amylose
content than what is observed in storage tissues.
16
The amylose structure is also
of a smaller molecular size.
1.1.2 Starch in storage tissues
In storage organ s, fruit or seed, during the development and maturation of the
tissue, synthesis of starch occurs. At the time of sprouting or germination of the
seed or tuber, or ripening of the fruit, starch degradation in thes e tissues then
occurs and the derived metabolites are used as a source both for carbon and
energy. The degradative and biosynthetic processes in the storage tissue s may
therefore be temporally separated. However, there is some possibility that during
each phase of starch metabolism some turnover of the starch molecule occurs.

The main site of starch synthesis and accumulation in the cereals is the
endosperm, with starch granules that are located within the amyloplasts. Starch
content in potato tube r, maize endosperm, and in roots of yam, cassava and
sweet potato ranges between 65 and 90% of the total dry matter. Patterns of
starch accumulation during development of the tissue are specific to the species
and are related to the unique pattern of differentiation of the organ.
Starch granules in storage tissues can vary in shape, size and composition.
The shape and size of the granules depends on the source, but in each tissue
there is a range of sizes and shapes. The diameter of the starch granule changes
during the development of the reserve tissue. There are also some fine features,
characteristic of each species, e.g., the `growth rings', spaced 4±7 "m apart, and
the fibrillar organ ization seen in potato sta rch, which allows one to identify the
botanical source of the starch by microscopic examination.
Two polymers are distinguished in the starch granule. Amylose, which is
essentially linear, and amylopectin, highly branched. Amylose is mainly found as
linear chains of about 840 to 22,000 units of -D-glucopyranosyl residues linked
by -(1->4) bonds (molecular weight around 136,000 to 3.5 Â 106). The number
ß 2004, Woodhead Publishing Limited
of anhydroglucose units, however, varies quite widely with plant species and stage
of development. Some of the amylose molecules are branched to a small extent
(-1->6-D glucopyranose; one per 170 to 500 glucosyl units). Amylopectin, in
contrast, which usually comprises about 70% of the starch granule, is more
highly branched with about 4 to 5% of the glucosidic linkages being -1->6.
Amylopectin molecules are large flattened disks consisting of -(1,4)-glucan
chains joined by frequent -(1,6)-branch points. Many models of amylopectin
structure have been proposed but from these the most satisfactory models, i.e.,
those that best fit the experimental data available, are those proposed by Robin
et al.
17
, Manners and Matheson

18
and by Hizukuri.
19
These are known as cluster
models. The chemical and physical aspects of the starch granule and its
components amylose and amylopectin have been discussed in some recent
excellent reviews by Morrison and Karkalis
20
and Hizukuri.
21
1.2 Starch synthesis: enzyme reactions in plants and algae
and glycogen synthesis in cyanobacteria
1.2.1 Enzyme reactions of starch synthesis
The sugar nucleotide utilized for synthesis of the -1,4 glucosidic linkages in
amylose and amylopectin is ADP-glucose and not UDP-glucose. ADP-glucose
synthesis is catalyzed by ADP-glucose (synthetase) pyrophosphorylase (reaction
1, E.C. 2.7.7.27; ATP:-D-glucose-1-phosphate adenylyltransferase).
ATP  -glucose-1-P @A ADP-glucose  PPi 1X1
ADP-glucose  -1,4 glucan A-1,4-glucosyl--1,4 glucan  ADP 1X2
Elongated -1,4-oligosaccharide chain A-1,4--1,6 branched-glucan 1X3
(pro-amylopectin) (phytoglycogen)
Reaction 2 is catalyzed by starch synthase (E.C. 2.4.1.21; ADP-glucose;1,4-
-D-glucan 4--glucosyltransferase). A similar reaction is noted for glycogen
synthesis in cyanobacteria and other bacteria (see references 22 and 23 but the
reaction is referred to as glycogen synthase (also E.C. 2.4.1.21). Reaction 3 is
catalyzed by branching enzyme (E.C. 2.4.1.18; 1,4--D-glucan 6--(1,4--
glucano)-transferase). The branch chains in amylopectin are longer (about 20 to
24 glucose units long) and there is less branching in amylopectin (~5% of the
glucosidic linkages are -1,6 as seen in glycogen (10±13 glucose units long and
10% of linkages are -1,6) . Thus, the starch branching enzymes may have

different properties with respect to size of chain transferred, or placement of
branch point, than enzyme that branches glycogen. Alternatively, the interaction
of the starch branching enzymes with the starch synthases may be different from
the interaction of the bacterial branching enzymes with their respective glycogen
synthases. The chain elongating properties of the starch synthases could be
different from those observed for the bacterial glycogen synthases and may
account for some of the differences observed in the amylopectin structure. The
ß 2004, Woodhead Publishing Limited
differences in the catal ytic properties of the starch synthases and branching
enzymes isolated from different plant sources may also account for the
differences observed in the various plant starch structures.
Isozymic forms of plant starch synthases (cited in references, 3, 4, 24±28) and
branching enzymes (cited in references 3, 4, 24, and in recent literature, 29±34)
have been reported. They seem to play different roles in the synthesis of the two
polymers of starch, amylose and amylopectin and are products from different
genes. In many different plants
35±40
as well as in Chlamydomonas reinhardtii
41
a granule-bound starch synthase involved in the catalysis of reaction (5) has
been shown to be involved in the synthesis of amylose. Mutants of many
different plants defective in this enzyme, are known as `waxy' mutants and give
rise to starch granules having only amylopectin.
Another enzyme, a debranching enzyme, most probably is involved in
synthesis of the starch granule and its polysaccharide components amylose and
amylopectin.
42±45
Soluble -glucan formed by reactions 1±3, is debranched to
form the amylopectin present in the starch granule
9, 46

and possibly provide a
primer in the starch granule for amylose synthesis by the granule-bound starch
synthase. The data strongly suggesting the role of a debranching enzyme in
synthesis of amylope ctin and the starch granule is discussed in a later section.
Reaction 2 was first described by Leloir et al.
47
with UDP-glucose as the
glycosyl donor, but it was later shown that ADP-glucose was more efficient in
terms of maximal velocity and km value.
48
Leaf starch synthases and the soluble
starch synthases of reserve tissues are specific for ADP-glucose. In contrast, the
starch synthases bound to the starch granule in reserve tissues do have some low
activity with UDP-glucose as compared to activity seen with ADP-glucose.
1.3 Properties of plant glucan synthesizing enzymes: ADP-
glucose pyrophosphorylase
1.3.1 Structure-function relationships
The ADP-glucose pyrophosphorylases (ADPGlc Ppase) of higher plants and
green algae, as well as the cyanobacteria, are proteins under allosteric control
and an important enzyme site for regulation of starch synthesis. The enzymes are
highly activated by 3-phosphoglycerate and inhibited by inorganic phosphate.
The regulation of starch and cyanobacterial glycogen synthesis via regulation of
the photosynthetic ADPGlc Ppase will be discussed in a later section. The
structural properties of the ADPGlc Ppase will be described in this section.
The kinetic and regulatory properties of the ADPGlc Ppases from the leaf
extracts of spinach, barley, butter lettuce, kidney bean, maize, peanut, rice,
sorghum, sugar beet, tobacco, and tomato have been studied in detail and are
similar.
49±52
The spinach leaf ADPGlc Ppase has been purified by either preparative disc gel

electrophoresis,
53
by hydrophobic chromatography
51
and the use of FPLC.
54
The
enzyme has a molecular mass of 206,000 and is composed of two different
subunits, of molecular mass es of 51 and 54 kDa.
54±57
These subunits are also
ß 2004, Woodhead Publishing Limited
distinguished with respect to amino acid composition, amino-terminal sequences,
peptide patterns of the tryptic digests on high-performance liquid chromatography
(HPLC), and antigenic properties. The two subunits are therefore quite distinct and
are the products of two different genes. The enzyme may be considered as having
an 22 structure. Bacterial ADPGlc Ppases including the cyanobacteria l
enzymes, in contrast, are homotetrameric, i.e., composed of only one subunit,
with a molecular mass of 50 to 55 kDa depending on the species.
58
Other plant ADPGlc Ppases have been shown to be composed of two
dissimilar subunits. The maize endosperm ADPGlc Ppase, which has a
molecular mass of 230 kDa, is composed of subunits of 55 and 60 kDa,
corresponding to the spinach leaf 51 and 54 kDa.
59
The maize endosperm mutants shrunken 2 (sh 2) and brittle 2 (bt2) are
ADPGlc Ppase activity deficient (reviewed references 3 and 4). In
immunoblotting experiments using antibodies raised against the native spinach
leaf enzyme and the individual subunits , it was found that the mutant bt2
endosperm lacks the 55 kDa subunit and the mutant sh 2 endosperm lacks the 60

kDa subunit. These results
59
strongly suggested that the maize endosperm
ADPGlc Ppase is composed of two immunologically distinctive subunits and
that the sh 2 and bt2 mutations cause reduction in ADPGlc Ppase activity
through the lack of one of the subunits; the sh 2 gene would be the structural
gene for the 60 kilodalton protein while the bt2 gene would be the structural
gene for the 55 kDa protein. Consistent with this hypothesis is the isolation of an
ADPGlc Ppase cDNA clone from a maize endosperm library
60
which hybridized
with the small subunit cDNA clon e from rice.
61
This maize ADP Glc Ppase
cDNA clone was found to hybridize to a transcript present in maize endosperm
but absent in bt2 endosperm. Thus, the bt2 mutant appears to be the structural
gene of the 55 kDa subunit of the ADPGlc Ppase.
Hylton and Smith
62
proposed the existence of not two, but four polypeptides
of MW around 50 kDa for the ADPglucose PPase of pea embryo, and a
molecular mass for the holoenzyme of about 110 kDa. The relationship of the
four subunits to the constitution of the native enzyme was not explained.
However, the available information of most systems indicates that both the seed
and leaf ADPglucose pyrophosphorylase are heterotetramers composed of two
different subunits, and that, on the basis of immunoreactivity and sequence
data,
63
there is close homology between the subunits in the leaf enzyme and with
the subunits of reserve tissue enzyme. Another point brought out by comparison

of the amino acid sequences of the the two different plant subunits with each
other and with the bacterial ADPGlc Ppase subunit amino acid sequences is that
the plant subunits may have evolved from the bacterial subunit.
63
1.3.2 Chemical modification of ligand binding sites of substrates and
effectors
Substrate sites
Because the plant native ADPGlc Ppases are tetrameric and composed of two
different subunits it was of interest to understand why two subunits are required
ß 2004, Woodhead Publishing Limited
for optimal catalytic activity in contrast to the bacterial ADPGlc Ppase. The
enzyme must contain ligand-binding sites for the activator, 3PGA, and inhibitor,
Pi, as well as catalytic sites for the two substrates, ATP and glucose-1-P, and it is
possible that these sites may be located on different subunits. Chemical
modification was used to obtain information on the catalytic mechanism and on
the catalytic site of the ADPGlc Ppase. Chemical modification studies on the
ADPGlc Ppase have involved the use of the following affinity labels: pyridoxal-
5-phosphate (PLP), an analogue of the activator 3-PGA as well as an analog of
the substrate, glucose-1-P; 8-azido-ATP and 8-azido-ADPGlc, photoaffinity
analogs of the substrates ATP and ADPGlc, respectively
64, 3
and phenylglyoxal,
for the identification of arginine residues that may be involved in substrate or
effector binding. These types of studies have provided information on the
catalytic and regulatory sites of the spinach ADPGlc Ppase and on the role of the
large and small subuni ts.
In studies with the E. coli ADPGlc Ppase, Lys residue 195 has been identified
as the binding site for the phosphate of glucose-1-P
65
and tyrosine residue 114

has been identified as involved in the binding of the adenosine portion of the
substrate, ATP.
66
The overall amino acid sequence identity of the E. coli
enzyme when aligned with the plant and cyanobact erial ADPGlc Ppases ranges
from 30 to 40%.
63, 67, 68, 69
However, there is greater sequence identity when the
E. coli ATP and glucose-1-P binding sites (Tables 1.1 and 1.2) are compared
with the corresponding sequences of the plant and cyanobacterial enzymes
suggesting that those sequences are still important in the plant enzyme, probably
having the same function. Indeed, in a recent preliminary experiment with the
potato tuber ADPGlc Ppase expressed in E. coli,
70
site-directed mutagenesis on
the lysine residue K198 of the 50kDa subunit (equivalent to the E. coli ADPGlc
Ppase K195 to a glutamate residue, increased the S0.5 value (concentration
required for 50% of maximal activity) for glucose-1-P from 57 "M to about
31 mM without any perceptible change on the km or Ka for the other substrates,
Mg+2, ATP or for activator, 3PGA (Y. Fu and J. Preiss, unpublished results,
Table 1.3). The apparent affinity of glucose-1-P was lowered over 500-fold.
Even a conservative mutation such as arginine replacing lysine at residue
198, caused a 135-fold decrease in the glucose- 1-P apparent affinity. These
results indicate an involvement of Lys residue 198 of the plant ADPGlc Ppase in
the binding of glucose-1-P. In the case of the putative ATP binding site instead
of tyrosine there is a phenylalanine residue in the corresponding sequences of
the plant and cyanobacterial enzymes (Table 1.2). It would be of interest to
determine in future site-directed mutagenesis and chemical modification studies
whether the WFQGTADAV region of the plant enzyme is indeed a portion of
the ATP binding region or whether the conservative change of two amino acids

in the sequence has affected the function of that portion of the protein. The
amino acids completely conserved are the tryptophan, glycine, threonine,
alanine and aspartate residues and possibly mutation of those residues would
indicate the relevan cy of this region as an ATP binding site.
ß 2004, Woodhead Publishing Limited
Activator sites
The binding site for pyridoxal phosphate in the spinach leaf ADPGlc Ppase small
subunit was isolated revealing a lysine residue close to the C-terminus which may
be important for 3PGA activation.
71
When PLP is covalently bound, the plant
ADPGlc Ppase is much less dependent on 3PGA for activation. The reductive
phosphopyridoxylation is also prevented by the allosteric effectors, 3PGA and Pi.
These observations showing that the modified enzyme no longer requires an
activator for high activity and that the covalent modification is prevented by the
presence of the allosteric effectors, strongly indicate that the activator analog, PLP,
is binding at the activator site. Ball and Preiss
72
showed also that three lysine
residues of the spinach leaf large subunit are also involved or close to the binding
site of pyridoxal-P and, presumably, of the activator, 3PGA. The chemical
modification of these Lys residues by pyridoxal-P was prevented by the presence
of 3PGA during the reductive phospho-pyridoxylation process and in the case of
the Lys residue of site 1 of the small subunit and site 2 of the large subunit (Table
Table 1.1 Conservation in plant ADPGlc Ppases of the Glucose-1-Phosphate (65) sites
present in E. coli ADPGlc PPase. References to these sequences for the plant ADPGlc
Ppases are in Smith-White and Preiss.
63
The sequences for the Anabaena enzyme is in
Charng et al.,

67
for the Synechocystis enzyme in Kakefuda et al.
68
and for the wheat
endosperm small subunit, in Ainsworth et al.
69
The number 195 corresponds to Lys195 of
the E. coli enzyme and | signifies the same amino acid as found in the E. coli enzyme
Source Glucose-1-P Site
PROKARYOTES
195
E. coli IIEFVEKP-AN
S. typhimurium ||D|||||-||
Anabaena V|D|S|||KGE
Synechocystis |TD|S|||QGE
PLANT SMALL SUBUNIT
A. thaliana leaf ||||A|||KGE
Barley endosperm ||||A|||KGE
Maize endosperm 54kDa ||||A|||KGE
Potato tuber 50kDa ||||A|||QGE
Rice seed |V||A|||KGE
Spinach leaf 51kDa ||||A|||KGE
Wheat endosperm ||||A|||KGE
PLANT LARGE SUBUNIT
A. thaliana leaf L|S|S|||KGD
Barley endosperm V|Q|S|||KGD
Maize endosperm 60 kDa VLQ|F|||KGA
Potato tuber 51kDa VVQ|A|||KGF
Spinach leaf 54kDa VLS|S|||KGD
Wheat endosperm (large subunit) VVQ|S|Q|KGD

1.4), Pi also prevented them from being modified by reductive pyridoxylation.
ß 2004, Woodhead Publishing Limited
Similar results were otained via reductive phosphopyridoxylation of the
Anabaena ADPGlc Ppase.
73
The modification was also prevented by 3PGA and Pi.
Lys419 was the modified residue and the adjacent sequences about that residue are
very similar to those observed for site 1 sequences of the higher plants (Table 1.4).
Site-directed mutagenesis of Lys 419 to either Arg, Ala, Gln, or Glu produced
mutant enzymes having 25 - to 150-fold lower apparent affinities, A0.5
(concentration of activator needed for 50% of maximal activation), than that of
wild-type enzyme (Table 1.5). No other kinetic constants such as affinity (km) for
substrates and the inhibitor, Pi, were affected. The heat stability or the catalytic
efficiency of the enzyme were also not affected. These mutant enzymes, however,
were still activated to a great extent at higher concentrations of 3PGA suggesting
that an additional site was involved in the binding of the activator. The Lys419Arg
mutant was chemically modified with the activator analog, PLP. Modification of
Lys382 in the Arg mutant was observed and caused a dramatic alteration in the
allosteric properties of the enzyme which could be prevented by the presence of
3PGA or Pi during the chemical modification process. Lys382 was thus identified
as another site involved in the binding of the activator and as seen in Table 1.4, the
sequence about Lys382 in the Anabaena enzyme is very similar to that seen for the
Table 1.2 Conservation in plant ADPGlc Ppases of the ATP binding sites present in E.
coli ADPGlc Ppase.
22
References to these sequences for the plant ADPGlc Ppases are in
Smith-White and Preiss.
63
The sequences for the Anabaena enzyme is in Charng et al.,
67

for the Synechocystis enzyme in Kakefuda et al.
68
and for the wheat endosperm small
subunit, in Ainsworth et al.
69
The number 114 corresponds to tyrosine of the E. coli
enzyme and | signifies the same amino acid as found in the E. coli enzyme
PROKARYOTES ATP Site
114
E. coli WYRGTADAV
S. typhimurium |||||||||
Anabaena |FQ||||||
Synechocystis |FQ||||||
PLANT SMALL SUBUNIT
A. thaliana leaf |FQ||||||
Barley endosperm |FQ||||||
Maize endosperm 54kDa |FQ||||||
Potato tuber 50kDa |FQ||||||
Rice seed |FQ||||||
Spinach leaf 51kDa |FQ||||||
Wheat endosperm |FQ||||||
PLANT LARGE SUBUNIT
A. thaliana leaf |FQ||||||
Barley endosperm |FR||||||
Maize endosperm 60 kDa |FQ||||SI
Potato tuber 51kDa |FQ||||||
Spinach leaf 54kDa | |FQ||||||
Wheat endosperm (large subunit) |FR|||||W
higher plants site 2 which is situated on the large subunit. Thus the site directed
ß 2004, Woodhead Publishing Limited

mutagenesis along with the reductive phosphopyridoxylation experiments strongly
indicate that in higher plants as well as in the cyanobacteria, lysine residues near
the carboxyl terminii of the ADPGlc Ppase subunits are part of the binding domain
of the allosteric activator.
Site 1 of the Anabaena enzyme corresponds to the lysyl residue near the C
terminus, Lys440, that is phospho pyridoxylated in the spinach leaf small
subunit,
71
and corresponds to Ly s468 in the rice seed small subunit and to
Lys441 in the potato tuber ADPGlc Ppase small subunit. Lys404 of the potato
tuber large subunit corresponds to Site 2 of the Anabaena enzyme, Lys382.
Table 1.3 The effect of site-directed mutagenesis of Lys residue 198 of the small
subunit of potato tuber ADPGlc Ppase on the Km of glucose-1-P. S refers to the 50 kDa
(small) subunit and L refers to the 51 kDa (large) subunit of the potato tuber enzyme. The
letters, K, R, A and E, are of the one alphabet code corresponding to the amino acids,
lysine, arginine, alanine and glutamate
Enzyme Km
(mM)
Wild-type (normal) 0.057
SK198R L-Wt 7.7
SK198A L-Wt 22.0
SK198E L-Wt 31.1
Table 1.4 Plant and cyanobacterial ADPglucose pyrophosphorylase activator binding
sites. The sequences are listed in one letter code and were taken from Smith White and
Preiss,
63
and from references indicated in the text. The Lys residues, covalently modified
by pyridoxal-P are in outline. The potato tuber enzyme Lys residue was identified via
site-directed mutagenesis. The numbers 419 and 382 correspond to the Lys residues in the
Anabaena ADPGlc Ppase subunit. Site 1 is present in the small subunit of the plant

ADPGlc Ppase while Site 2 is present in the large subunit
Activator Site 1 Activator Site 2
Cyanobacteria 419 382
Anabaena SGIVVVLKKNAVITDGTII QRRAIIDKKNAR
Synechocystis NGIVVVIKNVTIADGTVI IRRAIIDKNAR
Higher plants Activator Site 1, Activator Site 2,
(small subunit) (large subunit)
Arabidopsis thaliana SGIVTVIKDALIPTGVI IQECIIDKNAR
Barley endosperm SGIVTVIKDALLPSGTVI ISNCIIDMNAR
Maize endosperm GGIVTVIKDALLPSGTVI IRNCIIDMNAR
Potato tuber SGIVTVIKKDALIPSGIII IRKCIIDKNAK
Rice seed SGIVTVIKDALLLAEQLY INNCIIDMNAR
Spinach leaf SGIVTVIKKDALIPSGTVI IKDAIIDKKNAR
Wheat leaf IKRAIIDKNAR
Wheat seed SGIVTVIKDALLPSGTVI IQNCIIDKNAR
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