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First published online November 16, 2007; 10.1104/pp.107.109942 Plant Physiology 146:289-299 (2008) © 2008 American Society of Plant Biologists OPEN ACCESS ARTICLE
Heat Stability and Allosteric Properties of the Maize Endosperm ADP-Glucose Pyrophosphorylase Are Intimately Intertwined1,[OA]Program in Plant Molecular and Cellular Biology and Horticultural Sciences (S.K.B., J.R.S., L.C.H.), and Department of Chemistry (J.D.S.), University of Florida, Gainesville, Florida 32611–7200
ADP-glucose (Glc) pyrophosphorylase (AGPase), a key regulatory enzyme in starch biosynthesis, is highly regulated. Transgenic approaches in four plant species showed that alterations in either thermal stability or allosteric modulation increase starch synthesis. Here, we show that the classic regulators 3-phosphoglyceric acid (3-PGA) and inorganic phosphate (Pi) stabilize maize (Zea mays) endosperm AGPase to thermal inactivation. In addition, we show that glycerol phosphate and ribose-5-P increase the catalytic activity of maize AGPase to the same extent as the activator 3-PGA, albeit with higher Ka (activation constant) values. Activation by fructose-6-P and Glc-6-P is comparable to that of 3-PGA. The reactants ATP and ADP-Glc, but not Glc-1-P and pyrophosphate, protect AGPase from thermal inactivation, a result consistent with the ordered kinetic mechanism reported for other AGPases. 3-PGA acts synergistically with both ATP and ADP-Glc in heat protection, decreasing the substrate concentration needed for protection and increasing the extent of protection. Characterization of a series of activators and inhibitors suggests that they all bind at the same site or at mutually exclusive sites. Pi, the classic "inhibitor" of AGPase, binds to the enzyme in the absence of other metabolites, as determined by thermal protections experiments, but does not inhibit activity. Rather, Pi acts by displacing bound activators and returning the enzyme to its activity in their absence. Finally, we show from thermal inactivation studies that the enzyme exists in two forms that have significantly different stabilities and do not interconvert rapidly.
ADP-Glc pyrophosphorylase (AGPase) represents a rate-limiting step in starch synthesis in plants as well as glycogen biosynthesis in bacteria (for review, see Preiss and Romeo, 1994  ; Preiss and Sivak, 1996; Hannah, 2005). AGPase catalyzes the first committed step in the starch biosynthetic pathway, the conversion of ATP and  -Glc-1-P (G-1-P) to ADP-Glc and pyrophosphate (PPi; for review, see Hannah, 1997). Definitive evidence for the rate-limiting role of AGPase in starch synthesis has been derived through transgenic studies in potato (Solanum tuberosum) tubers (Stark et al., 1992) and seeds of wheat (Triticum aestivum; Smidansky et al., 2002), rice (Oryza sativa; Smidansky et al., 2003; Sakulsingharoja et al., 2004), and maize (Zea mays; Giroux et al., 1996; T.W. Greene and L.C. Hannah, unpublished data; Wang et al., 2007). Variants incorporated into plants giving rise to enhanced starch biosynthesis harbor alterations in allosteric properties and/or alterations in heat stability. Accordingly, the thermal stability and regulatory properties of AGPase have received considerable attention.
While bacterial AGPases are homotetramers, plant and unicellular green algae AGPases are composed of two identical small subunits and two identical large subunits (for review, see Georgelis et al., 2007 At least three mechanisms have been proposed to modulate plant AGPase activity: allosteric regulation, thermal inactivation, and reductive activation. Plant AGPases are activated by several allosteric effectors, with 3-phosphoglyceric acid (3-PGA) activation receiving the greatest attention. Inorganic phosphate (Pi) inhibits activity in many tissues. In the case of the maize endosperm AGPases, Pi negates 3-PGA activation and returns the enzyme's catalytic activity to its basal level observed in the absence of 3-PGA.
The thermal stability of AGPase is also an important characteristic because exposure to high temperatures during plant growth causes reduced grain yield in many internationally important cereal crops, including maize, wheat, and rice (for review, see Singletary et al., 1994
A Cys residue near the N terminus of the potato tuber small subunit (Fu et al., 1998
In the studies described below, we show that a diverse series of metabolites affects maize endosperm AGPase in two fundamentally different ways. Metabolites that serve as reactants, activators, or inhibitors of AGPase also stabilize activity to heat inactivation. This includes the reactants ADP-Glc and ATP as well as all known activators and inhibitors. We also describe two previously unknown activators of the maize endosperm AGPase. While the activators and inhibitors differ significantly in structure, all data are consistent with a common binding site (or mutually exclusive sites) for these effector molecules. Finally, we note that the enzyme can apparently exist in two states that differ in their rates of thermal denaturation. This observation has also been noted in apple (Malus domestica) leaf AGPase (Zhou and Cheng, 2005
Pi Binds Tightly to Maize Endosperm AGPase as Seen from Protection, But Does Not Inhibit Catalytic Activity
Allosteric regulation of AGPase is a physiologically important phenomenon. 3-PGA is the most characterized activator, and its activation is reduced by the presence of Pi. While this is a common feature of virtually all plant AGPases, interesting differences distinguish specific isoforms of the enzyme. In the case of the maize endosperm, Pi does not appreciably reduce activity in the absence of 3-PGA (Fig. 1
). Activity was measured in the ADP-Glc synthesis direction either in the presence or absence of 2.5 mM 3-PGA and at various concentrations of Pi. Very little Pi inhibition occurs if 3-PGA is omitted from the reaction mixture. This is in direct contrast to the closely related wheat endosperm AGPase (Gomez-Casati and Iglesias, 2002
Two mutually exclusive hypotheses explain Pi deactivation of the 3-PGA-stimulated maize endosperm AGPase: (1) Pi does not bind to this isoform in the absence of 3-PGA; or (2) Pi binding to AGPase occurs in the absence of 3-PGA, but binding has no appreciable effect on activity. To distinguish between these possibilities, we tested whether Pi, in the absence of 3-PGA, might affect other properties of maize endosperm AGPase. Heat stability was chosen because previous reports (Gomez-Casati et al., 2000 ; Zhou and Cheng, 2005) have shown that allosteric effectors and enzyme reactants enhance enzyme stability in the presence of elevated temperatures.
In the first series of experiments, we investigated whether the allosteric activator 3-PGA could reduce or negate heat-induced loss of maize endosperm AGPase activity (Fig. 2
). Purified maize endosperm AGPase was mixed with various concentrations of 3-PGA, and one aliquot of each mixture was incubated for 10 min at 42°C and then immediately placed on ice until assay. Heated and unheated aliquots of each mixture were then assayed for AGPase activity, and the percentage of remaining activity is presented in Figure 2. Clearly, 3-PGA reduces the heat lability of maize endosperm AGPase. This is consistent with reports of 3-PGA stabilization of other AGPases (Gomez-Casati et al., 2000
Pi, in the absence of 3-PGA, also stabilizes maize endosperm AGPase (Fig. 2). In fact, Pi provides substantially more protection than does 3-PGA at the concentrations shown (55% at 50 mM). Hence, the maize endosperm AGPase binds Pi in the absence of 3-PGA; however, this binding does not substantially reduce catalytic activity. To characterize Pi protection of maize endosperm AGPase in more detail, enzyme activity was measured following exposure to temperatures at 42°C for various times in the presence of several Pi concentrations (Fig. 3, A and B ). Activity loss follows first order kinetics with respect to time. Values of t1/2 (the time required for loss of one-half of the activity) at each Pi concentration were calculated from the slope of the plot of log activity versus time. t1/2 for inactivation increases with increasing amounts of Pi. A Kd of 65 µM ± 7 µM for Pi was then determined from a replot of the data using the method of Scrutton and Utter (1965) .
The Activators and Inhibitors of AGPase Are Intimately Intertwined
We next asked whether metabolites known or suspected (Ghosh and Preiss, 1966
We then tested the same series of metabolites for their ability to stabilize the enzyme to heat inactivation (Fig. 4 ). In addition to the activators listed, sulfate was also tested because sulfate inhibits the potato tuber homotetramer (Jin et al., 2005 ) and saturating levels of sulfate negate the activating effect of 3-PGA. The maize endosperm AGPase is also inhibited by SO4– with a Ki of approximately 20 mM with a [3-PGA] = 2.5 mM (S.K. Boehlein, unpublished data). As shown in Figure 4, all of the metabolites that activate or inhibit activation of the maize endosperm AGPase also stabilize the enzyme against thermal inactivation. While it is evident that all metabolites can stabilize enzyme activity, major differences are seen in terms of the extent of protection and the concentrations of metabolites required for maximal stabilization. For example, 3-PGA and SO4– protect the enzyme to the same extent and also require a similar concentration for maximal protection, while the amount of F-6-P has not reached saturation under these conditions. Pi provides the best protection at saturation against heat inactivation (Fig. 4). The reducing reagent, DTT, and a noneffector ion, KCl, had no effect on the heat stability of the enzyme (data not shown).
We next measured the ability of the substrates to protect AGPase activity. Purified AGPase was mixed with the substrates (5 mM final concentration) listed in Table II and exposed to 42°C for 7.5 min prior to assay in the forward (ADP-Glc synthesis) or back (ADP-Glc degradation) direction. Activities are presented as a percentage of activity remaining from a control maintained on ice prior to assays. Several effectors were also included in these studies. Various kinetic studies have shown that ATP is the first substrate bound to the enzyme and ADP-Glc is the last product released in other AGPase homologs (Paule and Preiss, 1971 ; Kleczkowski et al., 1993). Hence, it was expected that these kinetically productive complexes formed with reactants might prevent heat-induced activity loss. Under the conditions employed, ADP-Glc provided the greatest amount of protection against thermal inactivation (approximately 60%), regardless of direction. This protection was enhanced by adding either 3-PGA or Pi, and virtually complete protection under these experimental conditions was achieved by the addition of both ADP-Glc and 3-PGA (Fig. 5
). It is also interesting that 3-PGA greatly decreased the amount of ADP-Glc required for one-half maximal protection (Fig. 5). The synergistic effect was not surprising because 3-PGA decreases the Km for the substrates as well as increases Vmax (Boehlein et al., 2005).
Although ATP alone provided some protection (approximately 15%), the addition of Pi or 3-PGA to the ATP/enzyme mixture greatly enhanced enzyme stability at 42°C (Table II). Increasing MgCl2 and ATP concentration to 50 mM allowed 46% of the initial activity to be maintained after heat treatment. PPi did not protect the enzyme against thermal inactivation and actually reduced the amount of stabilization elicited by 3-PGA and Pi. G-1-P also was unable to stabilize the enzyme at concentrations up to 50 mM. Additional experiments were performed in which 3-PGA, ATP, and G-1-P were added to the enzyme in the presence of Ca2+. Ca2+ was used as a cofactor so ATP would bind, but the reaction would not proceed to an appreciable extent during the incubation period (Fu et al., 1998 ). This set of conditions produced no additional protection compared to experiments containing only ATP and 3-PGA. Also, the addition of ADP-Glc and PPi in the presence of Ca2+ did not condition additional protection compared to addition of only ADP-Glc (Table II). Taken together, these data are consistent with an ordered kinetic mechanism, with reactants binding productively to free enzyme. In turn, this conditions protection against heat-induced activity loss. 3-PGA and Pi protect against thermal inactivation independently and each acts synergistically with ADP-Glc and ATP to protect the enzyme. Substrates that do not bind productively to free enzyme (PPi and G-1-P) do not enhance thermal stability.
To further characterize binding of these effector molecules to AGPase, the method of Scrutton and Utter (1965)
We then probed the possibility that all the ligands responsible for modulating catalytic activity and thermal stability might bind to the site(s) occupied by 3-PGA. If so, several conditions should be met. First, the addition of a nonsaturating concentration of a second activator should enhance the activation caused by nonsaturating amounts of 3-PGA. This effect was observed when 0.1 mM 3-PGA (one-half Ka value) was added to the enzyme, followed by a second activator equal to one-half its Ka value. Significant additional stimulation was seen for most activators (Table IV ). A second prediction of this hypothesis is that a metabolite that activates to a lesser extent than 3-PGA should, at increasing concentrations, inhibit the activation produced by 3-PGA. Phosphoenolpyruvate (PEP) was chosen for this experiment because the activity in the presence of PEP was only about 40% of its activity in the presence of 3-PGA. 3-PGA was used at a concentration of 0.75 mM approximately (3 x Ka) and PEP was varied from 0 to 100 mM (0–20 x Ka). Inhibition by PEP begins at about 25 mM and increases up to about 100 mM (Fig. 6 ). The decrease in activity caused by the competition of activators indicates that PEP and 3-PGA are binding to the same site or to mutually exclusive sites.
Several observations suggest that Pi and 3-PGA share a common binding site. Heat protection studies were employed to further investigate this possibility. If 3-PGA and Pi bind to a common site, the presence of one will not have a synergistic effect on the other during thermal protection studies. If, on the other hand, binding of the activator and binding of the inhibitor are independent or coupled, a cumulative or synergistic pattern would be seen. To determine the relationship between 3-PGA and Pi, several activator/inhibitor combinations were used in thermal protection studies (Table II). First, when assayed in the reverse direction, 10 mM Pi conditions 64% protection under the conditions specified. Similarly, 3-PGA conditions 39% protection. Significantly, the combination of 3-PGA and Pi gives 67% protection, a value comparable to the protection observed with only Pi. This suggests that 3-PGA and Pi are binding to the same site or mutually exclusive binding sites. In contrast, we note that the addition of 3-PGA greatly increases the protection caused by the substrates ADP-Glc and ATP (Table II). This is indicative that 3-PGA (and Pi) and the substrates do not share common binding sites. Thus far, it has been shown that Pi can bind to the enzyme with a fairly high affinity; however, binding does not itself inhibit catalytic activity. A common binding site for Pi and 3-PGA should also be manifest by a Ki value for Pi that is dependent on 3-PGA concentration. This was observed (Fig. 7 ). As the 3-PGA concentration increases, the amount of Pi needed to inhibit activity rises. Thus, the role of Pi is not to inhibit the enzyme per se, but rather to deactivate the activated state by competing with the activator for binding to the enzyme. To determine whether Pi interacts with the effectors F-6-P and G-6-P in a similar manner, the Ki for Pi in the presence of 3-PGA, F-6-P, and G-6-P was determined. Enzymes were incubated with activators (3-PGA, F-6-P, or G-6-P) at 10x their corresponding Ka values. Pi inhibition studies were then performed to determine the Ki value of Pi in the presence of these activators. If phosphate is able to compete with these activators in a comparable manner, then all three of their phosphate Ki values should be the same. This was the case (Table V ).
Heat Denaturation Curves for AGPase Are Biphasic While all of the thermal protection experiments described above employed a temperature of 42°C and exposure times ranging up to 10 min, longer incubations at 42°C showed that catalytic activity remaining after the initial exposure to 7 to 10 min exhibited greater heat stability than the activity lost at the beginning of the experiment. Representative data are shown in Figure 8 . Catalytic activity was monitored over 40 min in the forward (Fig. 8A) and back (Fig. 8B) directions in the presence of 10 mM Pi. All plots exhibited a clear biphasic pattern of thermal inactivation with a break around 10 min. To determine whether the transition between the two states of the enzyme was a fast or slow process, AGPase plus Pi was placed at 42°C for 10 min and then cooled on ice for 5 min. The sample was then reincubated at 42°C and catalytic activity was monitored over time. If the transition back to the unstable form is a fast process, a rapid loss in enzyme activity should occur during the first 5 to 10 min following re-exposure to heat treatment. Conversely, if the transition is a slow process and the enzyme is locked into a more stable conformation, activity of the preheated enzyme should not exhibit a biphasic decay curve and a lower initial activity should be seen. As can be seen in Figure 8C, the preheated AGPase retains the properties of the more stable configuration of the enzyme. This experiment demonstrates that the enzyme form, which is rapidly heat inactivated, cannot be restored by cooling and that the remaining catalytic activity is due to a more heat-stable form to the enzyme. We then probed whether this interconversion of heat-labile to heat-stable form required the presence of Pi. At 42°C, the wild-type AGPase activity inactivates rapidly, in the absence of Pi, and a clear distinction between enzyme conformations was not obvious. The temperature of inactivation was therefore lowered to 37°C (Fig. 8D) and 30°C (data not shown). These data showed that the biphasic nature of decay is still present, thereby indicating that, although Pi protects the enzyme from heat inactivation, its presence is not obligatory for the biphasic loss of catalytic activity. A similar experiment was performed in the presence of DTT, and no discernable effect on the biphasic nature of inactivation was noted.
Plant transgenic approaches have shown that AGPases harboring alterations in allosteric properties as well as enhanced thermal stabilities can enhance starch synthesis in agriculturally important tissues and organs such as the potato tuber and seeds of maize, wheat, and rice. Accordingly, we have focused our investigations on these two properties. The results reported here show that allostery and heat stability are intimately connected. Metabolites that affect catalytic activity also affect heat stability. Several other features of the maize endosperm AGPase, as outlined below, are noteworthy. First, while 3-PGA has received the greatest attention as an activator of AGPase activity, we show that the maize endosperm AGPase can also be activated by a variety of other phosphate monoesters. Metabolites ranging from the three-carbon 3-PGA to the 12-carbon Suc-6-P cause comparable levels of activation. Activating metabolites appear to bind at the same or mutually exclusive sites in stimulating maize endosperm AGPase because the addition of metabolites to AGPase partially activated by 3-PGA result in further activity enhancements. This has been directly demonstrated for 3-PGA and PEP, and it is a distinct possibility that all activating metabolites bind to the same site.
While F-6-P and G-6-P both stimulate the activity of several AGPases (Ghosh and Preiss, 1965 We were surprised to find that glycerol phosphate stimulates the maize endosperm AGPase. This has not been reported for other AGPases, to the best of our knowledge. Whether this provides a mechanism to coordinate starch synthesis and glycolysis and/or lipid synthesis is presently unknown. Alternatively, this activation may simply reflect the structural similarity of glycerol phosphate and 3-PGA.
Metabolites that activate or inhibit AGPase also stabilize the enzyme to thermal inactivation. While Zhou and Cheng (2005)
Protection by ATP and ADP-Glc but not G-1-P and PPi against thermal inactivation is consistent with the well-precedented, ordered kinetic mechanism in which ATP is the first substrate bound and ADP-Glc is the last product released. In this regard, the maize endosperm AGPase exhibits a striking dissimilarity with a cyanobacterial AGPase. This bacterial enzyme is protected from thermal inactivation only by ATP (Gomez-Casati et al., 2000
The synergistic interaction of substrates and activators/inhibitors in protecting the maize endosperm AGPase against thermal inactivation suggests that enhanced heat protection occurs when both the active and allosteric sites are occupied. On the other hand, synergy was not observed in studies involving saturating levels of activators and Pi, suggesting that these species bind to the same or mutually exclusive sites. The data obtained from crystallization studies of the potato tuber small subunit homotetramer suggests that Pi has three or four separate binding sites on this particular enzyme and that 3-PGA binds at or near one of these inhibitor binding sites (Jin et al., 2005
Several explanations can account for the biphasic nature of the thermal stability exhibited by the maize endosperm AGPase. In the simplest case, two forms of enzyme exist at all times. The major form of activity, form 1, denatures rapidly, while the initially minor form 2 inactivates slowly. Therefore, during the first few minutes of heat treatment, large decreases in activity are seen from form 1 inactivation. This is then followed by a second, slow decrease in activity as form 2 is denatured. A second model assumes that before heat treatment, only one form of the enzyme exists. This form is heat labile. Exposure to heat causes the highly active, heat-labile form to convert to a more stable, less active form. Interconversion between the two forms is slow, giving rise to the observed biphasic pattern. A third, hybrid model can also be envisaged. Here, the enzyme exists as one heat-labile form. In the presence of heat, a proportion of molecules are denatured, whereas other molecules are slowly converted into a more heat-stable form. According to this model, both forms have comparable activity. The proposed interconversion of one form to another is reminiscent of the activation process of the potato tuber AGPase (Fu et al., 1998
Growth and Assay Conditions for Expression of Maize Wild-Type AGPase in Escherichia coli
Escherichia coli AC70R1-504 (Iglesias et al., 1993
Maize endosperm AGPase was purified as described elsewhere (Boehlein et al., 2005 Prior to kinetic analysis, proteins were desalted using Zeba micro desalt spin columns according to the manufacturer's instructions (Pierce). Proteins were routinely exchanged into 50 mM HEPES, 5 mM MgCl2, 0.5 mM EDTA, assayed for protein concentration, and then bovine serum albumin (BSA; 0.5 mg/mL) was added for stability.
Assay A (Reverse Direction)
Assay B (Forward Reaction)
Preparation of Coupling Reagent The coupling reagent contained a final concentration of 25 mM imidazole, pH 7.4, 4.0 mM MgCl2, 1.0 mM EDTA, 0.2 mM NADH, 0.725 units of aldolase, 0.4 units of triose phosphate isomerase, 0.6 units of glycerophosphate dehydrogenase, 1.0 mM F-6-P, and 0.8 µg of purified PPi-dependent phosphofructokinase per reaction. The coupling reagent was made fresh daily.
Determination of the Kinetic Constants
Heat Stability of Purified Maize AGPase
Sequence data from this article can be found in the GenBank/EMBL data libraries under accession numbers AF334959 and P55241. Received September 27, 2007; accepted November 7, 2007; published November 16, 2007.
1 This work was supported by the National Science Foundation (grant nos. IBN–9982626 and 0444031 to L.C.H.) and by the U.S. Department of Agriculture Competitive Grants Program (grant nos. 2000–01488, 2006–03034, and 2007–03575 to L.C.H.).  The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: L. Curtis Hannah (hannah{at}mail.ifas.ufl.edu).
[OA] Open Access articles can be viewed online without a subscription. www.plantphysiol.org/cgi/doi/10.1104/pp.107.109942 * Corresponding author; e-mail address hannah{at}mail.ifas.ufl.edu.
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ABSTRACT
RESULTS
) of 3-PGA or in the presence (
) of 2.5 mM 3-PGA is plotted.
), 1.0 (
), 2.5 (
), 5 (
), and 10 (
). Enzyme preparations were desalted and contained 0.5 mg/mL BSA. Various concentrations of Pi were then added prior to incubation on ice for 10 min. The samples were then heated at 42°C for various times. Duplicate samples were left on ice and their activity was taken to be 100%. All assays and calculations were determined as in 
