The sugar nucleotide utilized for synthesis of the (1 → 4) α -glucosidic linkages in amylose and amylopectin is ADP-glucose (ADPGlc). ADPGlc synthesis is catalyzed by ADP-glucose (synthase) pyrophosphorylase (ADPGlc PPase) (Reaction 4.1, E.C.
2.7.7.27; ATP: α -D-glucose-1-phosphate adenylyltransferase).
ATPα-glucose -P1 UADP-glucosePPi (4.1)
ADP-glucose(1→ α4)- -glucan→ → α(1 4)- -glucosy1-(1→ α4)- -glucanADP (4.2) Elongated ( )-linked malto-oligosacchar e chain
(
1 4
1 4 1 6
→
→ → →
id
, )) branched -glucanα (4.3)
Reaction 4.2 is catalyzed by starch synthase (E.C. 2.4.1.21; ADP-glucose: 1,4- α -D-glucan 4- α -glucosyltransferase). Similar reactions are noted for glycogen synthe-sis in cyanobacteria and other bacteria, 24 – 26 but the enzyme is referred to as glycogen synthase (also E.C. 2.4.1.21).
Reaction 4.3 is catalyzed by branching enzyme [E.C. 2.4.1.18; 1,4- α -D-glucan 6-α -(1,4- α -glucano)-transferase]. Amylopectin has longer chains ( ⬃ 20 – 24 glucosyl units) and has less branching [ ⬃ 5% of the α -glucosidic linkages are (l → 6)] com-pared to animal or bacterial glycogen [10 – 13 glucosyl units and 10% of linkages α -(l → 6)]. Thus, the plant branching enzymes may have different properties with respect to size of chain transferred, or placement of branch point, than the bacterial enzyme that produces glycogen. The differences in branching between glycogen and amylopectin may be explained by different interactions of branching enzyme with the different synthases. Possibly, the interaction of starch-branching isozymes with starch synthase isozymes may be different than the interaction of the bacterial glyco-gen synthase with the respective bacterial branching enzyme or the chain elongating properties of the starch synthases could be different from those observed for the bac-terial glycogen synthases. The differences in the catalytic properties of the different plant starch synthases and branching enzymes may also account for the variations observed in different plant starch polymer structures from different plants.
Isozymes of various plant starch synthases 2,3,27 – 31 and branching enzymes 2,3,27,32 – 37
have been reported. They are products of different genes and are proposed to play dif-ferent roles in the synthesis of amylopectin and amylose. In plants, 38 – 43 as well as in Chlamydomonas reinhardtii ,44 a granule-bound starch synthase involved in catalysis of Reaction 4.2 has been shown to be involved in the synthesis of amylose. Mutants of plants defective in this enzyme are known as waxy mutants, and give rise to starch granules having low amounts of or no amylose.
Another enzyme, a debranching enzyme, isoamylase, most probably is involved in synthesis of the starch granule and its polysaccharide components. 45 – 48 Mutant plants defi cient in isoamylase activity accumulate less starch granules and accumulate a soluble α -glucan termed phytoglycogen. 5,8,49 Data suggesting the role of a debranching enzyme in synthesis of amylopectin and the starch granule are discussed later (Section 4.12).
The starch synthase reaction was fi rst reported by Leloir et al. 49 with UDP-glucose (UDPGlc) as the glycosyl donor. It was later shown that ADP-glucose was much more effi cient in terms of maximal velocity and Km value. 51 Furthermore, leaf starch synthases and the soluble starch synthases of reserve tissues were later found to be specifi c for ADPGlc. In contrast, the granule-bound starch synthases in reserve tis-sues have ⬃ 1 – 10% activity with UDPGlc, as compared to the activity with ADPGlc.
The Km values for UDPGlc are usually in the mM range, while the Km values for ADPGlc are in theμ M range.
IV. Properties of the Plant 1,4- α -Glucan-Synthesizing Enzymes
1. ADP-glucose Pyrophosphorylase: Kinetic Properties and Quaternary Structure
The ADP-glucose pyrophosphorylases (ADPGlc PPase) of higher plants, green algae and the cyanobacteria are under allosteric control. The enzymes are highly activated by 3-phosphoglycerate (3PGA) and inhibited by inorganic orthophosphate (Pi), effects that are important for regulation of starch synthesis. The structural properties of the higher plant enzymes are discussed fi rst. The potato tuber 52 – 65 and spinach leaf ADPGlc PPases 66 – 74 have been studied in most detail with respect to kinetic proper-ties and structure. The kinetic and regulatory properproper-ties of the ADPGlc PPases from leaf extracts of barley, butter lettuce, kidney bean, maize, peanut, rice, sorghum, sugar beet, tobacco and tomato are similar to those of the spinach leaf enzyme ( Table 4.1 ). 69
Table 4.1 Kinetic constants from ADPGlc PPases from higher plants, green algae, and cyanobacteria
Source (reference) Effector Constant (mM) n Activation-fold Barley endosperm
purifi ed 75
ATP ( 3PGA) ATP ( 3PGA) G1P ( 3PGA) G1P ( 3PGA)
0.31 0.19 0.12 0.12
2.1 1.0 NR a NR
1.0
Barley leaves purifi ed 76
3PGA 0.005 1.0 20
Pi 0.025 1.0
ATP ( 3PGA) 1.0 1.0
ATP ( 3PGA) 0.08 1.0
G1P ( 3PGA) 0.33 1.0
G1P ( 3PGA) 0.11 1.0
Maize endosperm purifi ed 77
3PGA 0.12 1.0 25
3PGA ( Pi, 1 mM) 1.2 1.5 Pi ( 3PGA, 1 mM) 0.44 1.0
ATP ( 3PGA) 0.84 1.3
ATP ( 3PGA) 0.11 1.0
G1P ( 3PGA) 0.67 0.9
G1P ( 3PGA) 0.03 1.0
Tomato fruit 78,b 3PGA 0.2 NR Negligible
ATP ( 3PGA) 0.12 NR activity in
G1P ( 3PGA) 0.086 NR absence of
Pi (3PGA, 0.5 mM) 0.7 3PGA
Wheat endosperm79
Pi 0.7 1.3 No effect
3PGA No effect
3PGA ( Pi, 0.7 mM) 0.81 1.0 3PGA ( Pi, 1.5 mM) 1.51 1.4 3PGA ( Pi, 5 mM) 3.33 2.5
G1P 0.092 1.0
ATP 0.12 1.0
F6Pc No effect
F6Pc ( Pi, 0.7 mM) 2.5 NR
(continued)
IV. Properties of the Plant 1,4- α -Glucan-Synthesizing Enzymes 87
Table 4.1 (continued)
Source (reference) Effector Constant (mM) n Activation-fold
Wheat leaf 79 Pi 0.2 1.2 11
3PGA 0.01 1.0
3PGA ( Pi, 2.0 mM) 1.9 2.3
G1P 0.45 1.1
G1P ( 3PGA) 0.08 1.0
ATP 0.73 1.2
ATP ( 3PGA) 0.22 1.1
Chlamydomonas80 3PGA 0.23 1.3 15
Pi 0.054 1.0
Pi ( 3PGA, 2.5 mM)
0.53 1.7
G1P 0.22 1.7
G1P ( 3PGA) 0.03 1.2
ATP 0.48 1.2
ATP ( 3PGA) 0.08 1.3
Rice endosperm 81 3PGA 0.65 NR 40
Pi ( 3PGA, 1 mM) 0.40 NR
G1P ( 3PGA) 0.17 NR
ATP ( 3PGA) 0.18 NR
Arabidopsis 3PGA 0.34 NR NR
(recombinant APS1 APL1) d,82
3PGA ( Pi, 2 mM) 2.7 NR
G1P 0.06 NR
ATP 0.09 NR
Arabidopsis 3PGA 0.02 NR NR
(recombinant APS1 APL3) 83,d
Pi ( 3PGA, 1mM) 1.2 NR
G1P 0.20 NR
ATP 0.30 NR
Spinach leaf 68 3PGA 0.051 1.0 20
Pi 0.045 1.1
Pi ( 3PGA, 1 mM) 0.97 3.7
ATP 0.38 0.9
ATP ( 3PGA) 0.062 0.9
G1P 0.12 0.9
G1P ( 3PGA) 0.035 1.0
Potato tuber 56,58 3PGA 0.16 1.0 30
Pi ( 3PGA) 0.04 NR
Pi ( 3PGA, 3 mM) 0.63 NR
ATP ( 3PGA) 0.076 1.6
G1P ( 3PGA) 0.057 1.1
Anabaena84 3PGA 0.12 1.0 17
Pi ( 3PGA) 0.044 1.0
Pi ( 3PGA, 2.5 mM)
0.46 1.7
G1P ( 3PGA) 0.13 1.2
G1P ( 3PGA) 0.08 1.0
ATP ( 3PGA) 1.55 1.2
ATP ( 3PGA) 0.46 1.1
Synechocystis85 3PGA 0.81 2.0 126
Pi ( 3PGA) 0.095 1.0
(continued)
Table 4.1 (continued)
Source (reference) Effector Constant (mM) n Activation-fold Pi ( 3PGA,
2.5 mM)
0.57 2.2
G1P ( 3PGA) 0.18 1.1
G1P ( 3PGA) 0.05 1.1
ATP ( 3PGA) 3.2 2.2
ATP ( 3PGA) 0.80 1.0
a Not reported
b Negligible activity in the absence of 3PGA
c F6P D-fructose 6-phosphate
d Abbreviations: APSI, Arabidopsis small subunit. APL1 and APL3, Arabidopsis large subunits 1 and 3
The bacterial ADPGlc PPases are homotetrameric. 14 Thus, the catalytic sites, as well as the allosteric sites, reside on the same subunit. In plants and in green algae, however, the ADPGlc PPases are heterotetramers having two different, but homolo-gous, subunits, α2β2 , of different molecular sizes. 13,14,53,71,86 The small subunit has the catalytic activity and is ca. 50 – 54 kDa in size. The large subunit, which is the reg-ulatory subunit, having no catalytic activity, is approximately 51 – 60 kDa in size. 14,63 The large (regulatory) subunit modulates the sensitivity of the small subunit towards allosteric effectors via large subunit/small subunit interactions. 59
The ADPGlc PPase from potato tuber has been isolated and extensively character-ized.52,53,55,56 It is composed of two different subunits, 50 and 51 kDa in size and hav-ing anα2β2 heterotetrameric subunit structure. 53 Ballicora et al. 55 demonstrated that the small subunit of potato tuber ADPGlc PPase can be expressed as a homotetramer and is highly active in the presence of high concentrations of the activator 3PGA.
The large subunit could not be expressed in an active form, 56 suggesting that one subunit, the small subunit, can be designated as the catalytic subunit.
The small subunit of many higher plant ADPGlc PPases is highly conserved among plants, with 85 – 95% identity. 86 The structure of potato tuber ADPGlc PPase small subunit homotetramer is representive of that of higher plant enzymes. The homotetrameric potato enzyme, composed exclusively of small subunits, has a lower apparent affi nity (A 0.5 2.4 mM) for the activator, 3PGA, than the heterotetramer (A0.5 0.16 mM), and is also more sensitive to the inhibitor P i (I 0.5 0.08 mM in the presence of 3 mM 3PGA), as compared with the heterotetramer (I 0.5 0.63 mM). 56 The kinetic parameters of the homotetrameric small subunit appear to be non-physiological and thus, the non-physiological functional activity is due to the native enzyme in the α 2 β 2 structure containing both small (catalytic) and large (regula-tory) subunits.
In the case of potato tuber ADPGlc PPase, the large subunit greatly increases the affi nity of the small (catalytic) subunit for 3PGA and lowers its affi nity for the inhib-itor, Pi. 55,56 In a plant, there may be only one conserved small (catalytic) subunit and several large (regulatory) subunits that can be distributed in different parts of the plant.83,87 This is of physiological importance, as expression of different large subunits
IV. Properties of the Plant 1,4- α -Glucan-Synthesizing Enzymes 89
in different plant tissues may confer distinct allosteric properties to the ADPGlc PPase according to the different needs for starch synthesis in different tissues.
Results of this nature have been shown in the case of Arabidopsis ADPGlc Ppase. 83 Co-expression of its small subunit (APS1) with the different Arabidopsis large subu-nits (ApL1, ApL2, ApL3 and ApL4) resulted in heterotetramers with different reg-ulatory and kinetic properties ( Table 4.2 ). The heterotetramer of APS1 with ApL1, a large subunit predominant in leaves, 82,83 had the highest sensitivity to the allos-teric effectors 3PGA and Pi, as well as the highest apparent affi nity for the substrates ATP and glucose 1-phosphate (G1P). The heterotetrameric pairs of APS1 with either APL3 or APL4, large subunits more prevalent in sink or storage tissues, 87 had inter-mediate sensitivity to the allosteric effectors and interinter-mediate affi nity for the sub-strates ATP and G1P. 83 APL2, which is also present mainly in sink tissues, had very low affi nity for either 3PGA or Pi. 83 Thus, differences in the regulatory properties conferred by the Arabidopsis large subunits were found in vitro . Distinctions noted in the source and sink large subunit proteins strongly suggest that starch synthesis is modulated in response to 3PGA and Pi, as well as to the substrate levels, in a tissue-specifi c manner. APS1 and ApL1 would be fi nely regulated in source tissues by both effectors and substrates, while in sink tissues, the hetrotetramers of APS1 with APL2, APL3 or APL4, which have lower sensitivities to effectors and substrates, would be controlled more by the supply of substrates. 83
The pattern of expression and sugar regulation of the six Arabidopsis thaliana ADPGlc PPase-encoding genes (two small subunits, ApS1 and ApS2; and four large subunits, ApL1 – ApL4) have been studied. 87 Based on mRNA expression, ApS1 is the main small subunit or catalytic isoform responsible for ADPGlc PPase activity in all tissues of the plant. ApL1 is the main large subunit in source tissues whereas ApL3 and, to a lesser extent, ApL4 are the main isoforms present in sink tissues. It was also found that sugar regulation of ADPGlc PPase genes was restricted to ApL3
Table 4.2 Kinetic parameters for the 3PGA of A. thaliana recombinant ADPGlc PPase determined in the pyrophosphorolysis direction a,b
Control 0.2 mM Pi 2 mM Pi
3PGA A0.5, mM nH 3PGA A0.5, mM nH 3PGA A0.5, mM nH
APS1 d 1.2 0.09 1.8 5.6 0.29 2.5 ND c
APS1/APL1 0.0017 0.0005 1.0 0.019 0.0019 1.9 0.48 0.005 2.7
APS1/APL2 0.219 0.024 0.8 0.820 0.12 0.9 6.95 1.59 1.5
APS1/APL3 0.029 0.009 0.6 0.105 0.025 0.8 0.29 0.048 1.0 APS1/APL4 0.030 0.003 0.9 0.110 0.008 1.0 0.80 0.167 1.2
a The kinetic parameters were calculated without inhibitor (Pi) and in the presence of inhibitor at 0.2 mM or 2 mM
b The deviation in the 3PGA A0.5 data is the difference between duplicate experiments
c ND not determined
d APS1 is the Arabidopsis small (catalytic) subunit and APL1, APL2, APL3 and APL4, the Arabidopsis large subunits
and ApL4 in leaves. 87 Sucrose induction of ApL3 and ApL4 transcription in leaves allowed formation of heterotetramers less sensitive to the allosteric effectors, resem-bling the situation in sink tissues, which are regulated by an allosteric mechanism (3PGA/Pi ratio).