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Originally published In Press as doi:10.1074/jbc.M707447200 on January 16, 2008

J. Biol. Chem., Vol. 283, Issue 11, 6640-6647, March 14, 2008
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Direct Appraisal of the Potato Tuber ADP-glucose Pyrophosphorylase Large Subunit in Enzyme Function by Study of a Novel Mutant Form*Formula

Seon-Kap Hwang, Yasuko Nagai, Dongwook Kim, and Thomas W. Okita1

From the Institute of Biological Chemistry, Washington State University, Pullman, Washington 99164-6340

Received for publication, September 5, 2007 , and in revised form, January 8, 2008.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The higher plant ADP-glucose pyrophosphorylase is a heterotetramer consisting of two subunit types, which have evolved at different rates from a common ancestral gene. The potato tuber small subunit (SS) displays both catalytic and regulatory properties, whereas the exact role of the large subunit (LS), which contains substrate and effector binding sites, remains unresolved. We identified a mutation, S302N, which increased the solubility of the recombinant potato tuber LS and, in turn, enabling it to form a homotetrameric structure. The LS302N homotetramer possesses very little enzyme activity at a level 100-fold less than that seen for the unactivated SS homotetramer. Unlike the SS enzyme, however, the LS302N homotetramer enzyme is neither activated by the effector 3-phosphoglycerate nor inhibited by Pi. When combined with the catalytically silenced SS, SD143N, however, the LS302N-containing enzyme shows significantly enhanced catalytic activity and restored 3-PGA activation. This unmasking of catalytic and regulatory potential of the LS is conspicuously evident when the activities of the resurrected LK41R·T51K·S302N homotetramer are compared with its heterotetrameric form assembled with SD143N. Overall, these results indicate that the LS possesses catalytic and regulatory properties only when assembled with SS and that the net properties of the heterotetrameric enzyme is a product of subunit synergy.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
ADP-glucose pyrophosphorylase (AGPase, EC 2.7.7.2 [EC] 7)2 is a key enzyme in the control of starch synthesis in higher plants. The higher plant enzyme has a tetrameric structure consisting of two distinct subunit types, large subunit (LS) and small subunit (SS) (1–4). These subunit types share considerable sequence identity (~53%) and similarity (~73%) indicating that both subunits originated from a common gene ancestor which has diverged at different rates over time (5–7). When expressed in bacteria, the SS (SWT) of the potato tuber AGPase is capable of forming a catalytically active homotetrameric enzyme (8). Compared with the wild-type AGPase heterotetramer, the potato SWT homotetramer requires nearly 24-fold greater levels of 3-PGA for maximal enzyme activity and is more sensitive to the inhibitor inorganic phosphate (Pi) (8, 9). When fully activated by high concentrations of 3-PGA, the kinetic behavior of the SWT homotetramer is similar to the wild-type heterotetramer and cyanobacterial AGPases (10). These observations indicate that the LS is essential for optimal allosteric regulatory properties of the heterotetrameric enzyme. It is also noteworthy that introduction of only one or two amino acid changes generates a SS homotetramer with regulatory properties that are comparable to or more sensitive to 3-PGA than the wild-type heterotetrameric enzymes (8, 11).

Earlier studies (12, 13) suggested that the inter-subunit disulfide bond between two SSs of the potato tuber AGPase heterotetramer also plays a significant role in controlling enzyme activity and stability. When the bond was cleaved by reduction of cysteine residues by DTT or thioredoxins, the AGPase became more sensitive to activation by low levels of 3-PGA and less stable at elevating temperature. This redox modification of the plant AGPase was more evident in planta and significantly affected AGPase activities and starch synthesis (14–17).

By contrast, less information is available on the role of the LS subunit in enzyme function. Unlike the SS, the potato tuber AGPase LS is unable to self-assemble into an active tetramer when expressed in bacteria and readily forms inclusion bodies. Hence, all available evidence on the role of LS for the potato tuber AGPase activity has been obtained by studying its operability with the SS. Site-directed mutagenesis studies on conserved residues predicted to bind to effectors and substrates in the potato tuber subunits, suggest that the SS is catalytic and regulatory, whereas the LS lacked these properties and simply modulates the regulatory activity of the SS by protein-to-protein interactions (18, 19). An alternative view on subunit roles in enzyme function was obtained by the study of potato heterotetrameric enzymes formed by different combinations of wild-type and mutant LSs and SSs (9). Such results showed that the net allosteric properties of the heterotetrameric enzyme is contributed by both subunits and is a product of synergy between LS and SS interactions. A similar conclusion for a regulatory role for the LS was also made for the maize endosperm AGPase (20).

The LS of the potato tuber AGPase is also likely to significantly participate in catalysis by affecting the capacity of the heterotetrameric enzyme to bind substrates and effectors (6). Substantial evidence for a catalytic role, albeit indirect, for the LS was obtained by identification of a substrate binding site for ATP by photoaffinity labeling studies with 8-N3-ATP (21). Mutations of selected residues located within ATP binding site of the LS significantly altered the catalytic properties of the AGPase heterotetramer.

Based on homology studies of other structurally related sugar nucleotide pyrophosphorylases (22–24), the metal-binding Asp143 (originally assigned as Asp145) of the potato AGPase SS was suggested to be essential for catalytic activity, a prediction verified by site-directed mutagenesis of this residue, which lowered catalytic rate more than four orders of magnitude compared with the wild-type enzyme. A similar mutation in the conserved Asp158 of the LS, however, reduced catalysis only 1.5- to 2.6-fold (19). Our recent result, however, showed that the replacement of Asp158 by Leu lowered catalytic rates by 11-fold and catalytic efficiencies by 23- to 26-fold depending on the substrates of the enzyme (21), indicating the Asp158 residue in the LS is important for enzyme catalysis. Interestingly, introduction of a single residue replacement, T51K, or double mutations, K41R and T51K, into the wild-type LS resurrected partially (5% of wild-type activity) the catalytic activity of the heterotetrameric enzyme containing the catalytic silenced SD143N (7). Overall, these results, together with those obtained from earlier studies on substrate and effector binding, indicate that the LS is catalytically inefficient but has many of the necessary elements for this function.

Although available evidence indicates that the LS, which is capable of binding substrates (6, 18) and effectors (25), is catalytically defective, direct evidence for this property has yet to be obtained as all available evidence in support of this view is deduced from studies of the heterotetrameric enzyme forms. To further enhance our understanding of the role of LS in enzyme function, we identified a mutant LS containing a S302N replacement, which significantly elevated solubility of the LS and enabling assembly and formation of LS homotetramer. Results from biochemical and kinetic analysis of LS homotetramer and heterotetrameric forms with catalytic-silenced SD143N showed that the LS in the absence of SS displays very low catalytic activity and is allosteric-insensitive. When operating with the SS, however, catalysis by the LS is stimulated, and allosteric regulatory properties of the LS are unmasked.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Materials—[14C]Glucose 1-phosphate and [32P]pyrophosphate were purchased from ICN Pharmaceuticals and PerkinElmer Life Sciences, respectively. Radioactive 8-azidoadenosine 5'-[{alpha}-32P]triphosphate ([{alpha}-32P]8-N3-ATP) and non-radioactive 8-N3-ATP were purchased from Affinity Labeling Technologies, Inc. Reagents including ATP, Glc 1-P, and ADP-glucose were obtained from the Sigma-Aldrich and were of analytical grade or higher.

Expression and Purification of the AGPase Proteins—The wild-type and various LS mutants were expressed in the absence or presence of wild-type SS (SWT) or catalytic-silenced D143N SS (SSi) in the Escherichia coli EA345 host strain, which contains a null mutation in the AGPase gene glgC (6). The plasmids pSH274 and pSH208 (6) were used for expression of the His6-tagged LS and SS of the potato tuber AGPase, respectively. For high throughput expression of AGPase subunits E. coli EA3457 cell was made by transforming the pRARE plasmid isolated from RossetaTM cell (Novagen) into EA345 cell. For purification of the SS homotetramer the His6-tagged SWT protein (26) was expressed in EA3457 cells. AGPases were purified as described previously using a Bio-Logic DuoFlow Chromatography system (Bio-Rad) (6, 21) with minor modifications. Briefly, the active AGPase fractions obtained from a DEAE-Sepharose FF (Amersham Biosciences) chromatography and TALONTM-IMAC (Clontech Lab) were subjected to POROS 20 HQ (PerSeptive Biosystems) column chromatography. Aliquots of the purified enzyme preparation were stored frozen in liquid nitrogen and shelved at –80 °C until used for analysis.

AGPase Assay and Kinetics—AGPase activities were assayed in the pyrophosphorylase direction (Assay A) during enzyme purification and in the ADP-glucose synthesis direction (Assay B) for kinetic characterization (6). Unless stated, saturating amount of substrates was used for both methods. One enzyme unit is defined as 1 micromole of ATP (Assay A) or ADP-glucose (Assay B) formed for 1 min at 37 °C. KaleidaGraph 3.5 (Synergy software) was used to fit the experimental data to the modified Hill equations (6). The kinetic values (S0.5, A0.5, kcat, and kcat/S0.5) were determined as described previously (6).

Chemical and Site-directed Mutagenesis—200 µg of plasmid DNA (pSH345 or pSH274 containing LK41R·T51K) were subjected to chemical mutagenesis by incubating at 37 °C for 20 h in 4 ml of 0.1 M sodium phosphate (pH 6.0), 0.8 M hydroxylamine-HCl, and 1 mM EDTA (27). After neutralization with 400 µl of 1.5 M Tris-HCl (pH 8.8) the plasmid DNAs were then precipitated in 80% (v/v) ethanol. The plasmid DNAs were then used to transform E. coli EA345 cells expressing SSi. Cells were grown on NZCYM media supplemented with 100 mg/liter ampicillin and 50 mg/liter kanamycin with 0.4% (v/v) glycerol as the carbon source, which readily allows the efficient induced expression of both subunits from the lac promoters (6). After overnight culture at 37 °C, the bacterial colonies were screened for glycogen production by exposure to iodine vapor. Plasmid DNAs expressing the LS mutants were purified and were subjected to DNA sequencing to identify changes in nucleotide sequence.

Site-directed mutagenesis of the AGPase subunit sequence was accomplished using the QuikChange site-directed mutagenesis kit (Stratagene) as described previously (21): LS302N-F, 5'-AAATCGTTTTATAATGCTaacTTGGCACTCACACAAGAG-3' and LS302N-R, 5'-CTCTTGTGTGAGTGCCAAgttAGCATTATAAAACGATTT-3' (target sequences are in lowercase).

Labeling of AGPases with 8-Azido-adenosine 5'-Triphosphate—The purified AGPases were reduced, desalted, and labeled as described previously (6, 21).

Modeling of AGPase Structures—DeepView, the Swiss-Pdb-Viewer was used for modeling of the three-dimensional structure of the LS based on the crystal structure of the potato SS homotetramer (28) as the template scaffold. Coordinates for the AGPase structures (1yp3) were retrieved from RCSB Protein Data Bank. Pov-Ray was used for rendering the structure.


Figure 1
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  		FIGURE 1.
Purification profiles of various AGPases. AGPase proteins were initially purified on DEAE-Sepharose FF chromatography followed by TALONTM-immobilized affinity chromatography. The resulting enzyme preparation was then loaded onto a POROS 20HQ column (HR-5/5, bed volume, 1 ml) and washed with 0.15 M NaCl and eluted with 0.3 M NaCl (A) at the flow rate of 2 ml/min. Fractions containing proteins were analyzed by SDS-PAGE and immunoblot analysis (B) using antibodies raised against the potato LS (53 kDa) or SS (50 kDa). For convenience abbreviations were used for various mutations: R for K41R; K for T51K; N for S302N; and Si for D143N. CBB, Coomassie Brilliant Blue R-250.

 
Protein Analysis—Protein concentration was measured using the Advanced Protein Assay Reagent from Cytoskeleton (Denver, CO) with bovine serum albumin (fraction V) as the standard. SDS-PAGE was performed as described in a previous study (26). Immunoblot analysis was performed using anti-potato AGPase LS or anti-potato AGPase SS as described before (6).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Substitution of Ser302 with Asn in the LS Significantly Enhanced Glycogen Production in Bacterial Host Cells—To provide further insights on the role of the potato tuber LS in AGPase function, the expression plasmid DNA containing His6-tagged LK41R·T51K (abbreviated LRK) was subjected to chemical mutagenesis (supplemental Fig. S1A) and co-expressed with the catalytic-silenced SD143N (abbreviated SSi) mutant (7, 21) in bacterial cells lacking AGPase activity. These cells were then analyzed for glycogen production by exposure to iodine vapor. Under these conditions, cell expressing LWTSSi are devoid of glycogen and do not stain, whereas cells expressing LRKSSi enzyme stain lightly as they accumulate small but readily detectable levels of glycogen due to the low catalytic activity of this enzyme (supplemental Fig. S1B). More than 6 x 104 bacterial colonies derived from the mutagenized potato tuber AGPase LS were examined by iodine staining resulting in the identification of 26 colonies, which exhibited significantly enhanced glycogen accumulation (supplemental Fig. S1B). Interestingly, sequence analysis showed that all 26 LS sequences obtained from these excess glycogen accumulating cells contained a common third mutation, S302N, in addition to the pre-existing K41R and T51K mutations (for brevity, LS containing these three mutations will be denoted as LRKN). Six plasmids contained silent mutations in addition to S302N, whereas a single plasmid contained a fourth mutation V159I in LS. Thus, addition of S302N mutation in the LS enhances net AGPase activity which, in turn, increases glycogen production.

The S302N Mutation Dramatically Increases LS Solubility—Heterotetrameric forms of the LRKSSi and mutant LRKNSSi enzymes were purified to near homogeneity (>95%) by multiple chromatography steps. During the final clean-up of the enzyme activity using a strong anion-exchange chromatography step, an anomalous protein elution profile was observed for the LRKNSSi enzyme. Typically, the wild-type AGPase elutes from the anion-exchange column as a single major protein peak at 0.3 M NaCl with very little protein eluting at lower salt concentrations (21). Similar elution profile was also observed for the LRKSSi enzyme (Fig. 1A). Although much of the LRKNSSi enzyme also eluted at 0.3 M NaCl, a prominent peak was also observed eluting at 0.15 M NaCl (peak a of Fig. 1A, lower panel). Results from SDS-PAGE and immunoblot analysis showed that the 0.15 M NaCl fraction contained only the LS (Fig. 1B), indicating that a significant amount of LRKN was unassembled with the SS and remained soluble throughout the enzyme purification steps. Analysis of the 0.15 M NaCl protein fraction by Superdex-200 gel filtration chromatography indicated that the LRKN eluted with a molecular size of 223 kDa (supplemental Fig. S2). Hence, the soluble LRKN readily self-assembles into a homotetrameric form. Further studies showed that the soluble LN also readily self-assembles into a 220 kDa oligomer (supplemental Fig. S2).

S302N Mutation Is Responsible for the Enhanced Solubility and Formation of the Potato LS Homotetramer—To determine whether the Asn302 residue was responsible for the increase in solubility of the potato tuber LS we compared the total and soluble expression of various LSs containing or lacking this mutation. When expressed in E. coli cells alone without the SS counterpart, all of the various LS types were expressed at nearly equal levels when total cell extracts were examined by SDS-PAGE (supplemental Fig. S3A). When only the soluble fractions were examined, however, only the LS forms containing S302N were found at substantial levels while the same LS forms lacking S302N were present at very low levels indicating that the bulk of the expressed LS was insoluble (supplemental Fig. S3, B and C). Quantitative analysis of the soluble forms of the homotetramers after enzyme purification showed that LRKN and LN were 376- and 77-fold more soluble than LKR and wild-type L (LWT), respectively (supplemental Table S1). These observations indicate that introduction of an Asn residue at position 302 is responsible for the enhanced solubility of the potato tuber LS.

The LS Homotetramer Is Not Affected by Allosteric Effectors—Examination of the enzymatic activity of purified LN homotetrameric protein showed that it had very low but measurable activity (~0.4 unit/g). This activity was 93-fold less than that measured for the homotetramer SWT (37 units/g) when assayed in the absence of the activator 3-PGA (Table 1). AGPase activity was elevated >800-fold by the introduction of K41R and T51K substitutions into LN to yield LRKN (330 units/g).


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  		TABLE 1
Regulatory properties of the AGPases

Enzyme reactions were performed at saturating concentrations of substrates: 20 mM Glc-1-P and 5 mM ATP for LRKN and LN; 2 mM Glc-1-P and 2 mM ATP for the rest of the enzymes. For assay of the enzymes with very low enzyme activities, the amount of purified protein used for assay was increased (3.8 µg for LWTSSi, 17.1 µg for LN, 7.2 µg for LNSSi, or 12.9 µg for SSi). Depicted are the mean values (±S.E.) of at least two independent experiments. The A0.5 value corresponds to the 3-PGA concentration (µM) required for 50% maximal activation of enzyme. nH, Hill coefficient. Superscripted abbreviations are used for various mutations: WT for wildtype, R for K41R, K for T51K, N for S302N, and Si for D143N.

 
To determine whether the relatively low catalytic activity of LN and LRKN homotetramers was due to its dependence on allosteric activation, the enzyme activity was assayed over a broad range of 3-PGA concentrations. Interestingly, unlike the allosteric behavior of the homotetrameric SWT, which requires nanomolar levels of 3-PGA for maximum activation, the catalytic activity of LRKN (or LN) was not affected by low amounts of 3-PGA and was slightly inhibited at 3-PGA levels of >1 mM (Fig. 2). Likewise, LRKN homotetramer activity was not affected by the metabolic inhibitor, Pi, in the presence of 0.5 mM 3-PGA (Fig. 3). Hence, these results indicate that the LRKN homotetramer is not allosterically regulated by 3-PGA or Pi. Other metabolites (0.5 mM each of 2-PGA, phosphoenol pyruvate, fructose 6-phosphate, fructose 1,6-diphosphate, and AMP) tested showed no discernible effects on enzyme activity of the LS homotetramer (supplemental Fig. S4).


Figure 2
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  		FIGURE 2.
3-PGA activation profiles of the AGPase wild-type and mutants. Reaction was performed in the forward (ADP-glucose synthesis) direction at various 3-PGA concentrations under saturating substrate conditions (A–F) (see legend of Table 1). The experimental data were analyzed using modified Hill equation (6) with the curve fitting software, KaleidaGraph 3.5. A, LWTSWT; B, LRKNSWT; C, LNSWT; D, SWT; E, LRKN; and F, LN.

 
In addition to the homotetrameric enzymes, we also evaluated the potential allosteric regulatory properties of these LS types when assembled with SSi. Interestingly, unlike the LN and LRKN homotetramers, which are not subject to allosteric activation by 3-PGA, LNSSi, LRKSSi, and LRKNSSi readily respond to this effector. Activity of LNSSi increased 30-fold in the presence of 3-PGA. LRKSSi and LRKNSSi showed a different activity pattern in the absence and presence of 3-PGA. Without 3-PGA, the activities of the heterotetramers LRKSSi and LRKNSSi were significantly lower (5- and 7-fold, respectively) than the LRKN homotetramer, whereas the presence of 3-PGA mediated a 51- to 71-fold increases in enzyme activities of these heterotetramers.

The LS Mutant Homotetramer Is Catalytically Very Inefficient—The LRKN homotetramer showed significantly low affinity toward ATP and Glc 1-P compared with the heterotetrameric enzyme forms (Table 2). The S0.5 values for ATP were 1927 µM (nH = 1.9) in the presence of 0.5 mM 3-PGA and 1880 µM (nH = 1.6) in the absence of 3-PGA. The S0.5 values for Glc 1-P were 4307 or 4175 µM (nH = 1.3 or 1.4) in the presence or absence of 0.5 mM 3-PGA. When kinetic study was done in the pyrophosphorylase direction, similar trends in S0.5 values were obtained for ADP-glucose and PPi with the LRKN homotetramer having S0.5 values of 3- to 5-fold and 2- to 3-fold higher than LRKNSWT, respectively (Table 3). These results also indicate that the catalytic properties of the LS mutant are not affected by 3-PGA, and the LS mutant is less efficient at substrate binding than the enzyme forms containing SS.


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  		TABLE 2
Catalytic properties of the various AGPases in the ADP-glucose synthesis (forward) direction

Kinetic parameters were determined in the presence of saturating concentrations of substrates: 15 mM Glc-1-P and 5 mM ATP for LRKN homotetramer, and 2 mM Glc-1-P and 2 mM ATP for the rest of the nzymes. Concentrations of 3-PGA were 0.5 mM for the LRKN homotetramer, 5 mM for the various AGPase heterotetramers, and 10 mM for SWT homotetramer. Depicted are the mean values (±S.E.) obtained from at least two independent experiments. The S0.5 value corresponds to the substrate concentration (µM) required for 50% maximal activity of enzyme. nH, Hill coefficient. C.E.: catalytic efficiency (kcat/S0.5), x 103.

 


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  		TABLE 3
Catalytic properties of the various AGPases

Activity was determined in the reverse pyrophosphorolysis direction in the presence of saturating concentrations of substrates: 2 mM pyrophosphate and 10 mM ADP-glucose for LRKN homotetramer, and 1.5 mM pyrophosphate and 2 mM ADP-glucose for the rest of the enzymes. Concentrations of 3-PGA used in the assays were 0.5 mM for LRKN homotetramer, 5 mM for the various AGPase heterotetramers, and 10 mM for SWT homotetramer. The S0.5 value corresponds to the substrate concentration (µM) required for 50% maximal activity of enzyme. Presented are the mean values (±S.E.) obtained from at least two independent experiments. nH, Hill coefficient. C.E.: catalytic efficiency (kcat/S0.5), x 103.

 
The LS Mutant Homotetramer Binds ATP But Less Efficiently—Our previous studies (6, 21) have demonstrated that both the LS and SS in the heterotetrameric form are labeled at equivalent rates with 8-N3-ATP, an analog which can readily substitute for ATP in catalysis. To determine the LS binding properties for ATP, LRKN and LN homotetramers were incubated with increasing concentrations of radioactive 8-N3-ATP analog (Fig. 4) using LRKNSWT and SWT as the controls. The labeling patterns obtained showed that irrespective of enzyme activity (LRKN = 330 units/g and LN = 0.4 unit/g) both LS homotetramers were photoaffinity-labeled with the ATP analog at similar efficiencies: ATP labeling constants (KL) for LRKN and LN were estimated to be 590 µM and 530 µM, respectively. The labeling constant for LRKN was 3.3- and 2.5-fold higher than those obtained for the SWT homotetramer (KL = 180 µM) and LRKNSWT heterotetramer (KL = 230 µM), respectively. Moreover, unlike the similar affinity values obtained by photoaffinity labeling (KL) and enzyme kinetics (S0.5) obtained for the SWT and LRKNSWT forms, the KL values of the LS homotetramers were ~3- to 4-fold lower than the apparent ATP S0.5 for LRKN. This significant disparity indicates that the apparent S0.5 for ATP for the LRKN homotetramer overestimates the actual binding efficiency of ATP likely due to its poor catalytic activity by the LS. Moreover, it demonstrates that the K41R and T51K replacements alter the catalytic rate and not the substrate binding properties.


Figure 3
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  		FIGURE 3.
Phosphate inhibition profiles of the AGPases. Reactions were performed in the forward (ADP-glucose synthesis) direction under saturating substrate conditions and with 0.5 mM 3-PGA (see legend of Table 1). The smooth fit feature of KaleidaGraph 3.5 was used for curve fitting. LRKN ({triangleup}), SWT ({square}), and LRKNSWT ({circ}).

 
The LS Mutant Homotetramer Is Less Heat-stable—Heat stabilities of the LS mutant homotetramer (LRKN), SS homotetramer (SWT), and their combined heterotetramer (LRKNSWT) were examined by incubating the enzymes at 30 °C, 37 °C, 45 °C, 52 °C, and 60 °C for 5 min. The enzyme activity remaining was measured at saturated concentrations of substrates and 3-PGA (Fig. 5). At 60 °C the LRKNSWT heterotetramer retained 92% of AGPase activity and the SWT homotetramer had a intermediate level of activity (68%), whereas the LRKN showed only <7% of activity. This result indicates that the homotetrameric form of the potato tuber LS is more susceptible to heat denaturation compared with SWT and LRKNSWT heterotetramer.


Figure 4
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  		FIGURE 4.
Photoaffinity labeling of the LS mutants by 8-N3-ATP. A, purified LRKNSWT, SWT, LRKN, and LN (1.5 µg each) were labeled with 0–1.6 mM of [{alpha}-32P]8-N3-ATP in 50 mM Hepes (pH 7.0), 7 mM MgCl2, and either 10 mM 3-PGA for LWTSWT and SWT or 1 mM 3-PGA for LN and LRKN in the absence of Glc 1-P. The proteins were then separated by electrophoresis on 10% SDS-polyacrylamide gels. The gel was stained with CBB R-250, dried, and autoradiographed. B, the ATP binding (labeling) curves. The labeling curve was plotted with KaleidaGraph 3.5 by using a modified Scatchard equation (6).

 


Figure 5
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  		FIGURE 5.
Thermal stability of the LS homotetramer. Samples of LRKNSWT, SWT, and LRKN (1.5 µg each) in 40 µl of H buffer (50 mM HEPES, pH 7.0, 10 mM MgCl2, 200 mM NaCl, 10% v/v glycerol, 2 mg/ml bovine serum albumin) were heat-treated at 30 °C, 37 °C, 45 °C, 52 °C, and 60 °C for 5 min and then immediately placed on ice. Enzyme activity was measured after diluting the protein samples with 50 mM HEPES-NaOH (pH 7.0) and 3 mM DTT and incubating at room temperature for 30 min. Relative activity (%) was calculated with respect to the activity of the samples previously placed on ice. The polynomial fit (order = 3) of KaleidaGraph 3.5 was used for curve fitting.

 
The LS Mutant Homotetramer Is Not Redox-regulated—Under non-reducing conditions, the various heterotetrameric AGPases, including wild-type LWTSWT (Fig. 7A) and mutants LRKNSWT and LNSWT (Fig. 7, B and C, respectively) showed that a significant proportion of the SSs were linked by an interchain disulfide bond. This condition was also the case for the SWT homotetramer (Fig. 7D). Treatment of these enzymes with DTT resulted in the disappearance of the SS dimer (~100 kDa). Under the same conditions, the LS remained as a single subunit in the LN and LRKN homotetramers (Fig. 7, E and F).

We also examined the effects of the reducing agent (DTT) together with ADP-glucose on the regulatory properties of various AGPase heterotetramers and homotetramers (supplemental Table S2 and Fig. S5). Consistent with results from previous reports (12, 17), the reduced form of the wild-type AGPase heterotetramer showed higher activation at lower concentrations of 3-PGA than its non-reduced form. The same trends were observed for the other mutant heterotetramers. The increased activation was most remarkable for the SWT homotetramer. However, the reductive activation was not observed for LRKN homotetramer. In addition, the substitutions (K41R, T51K, and/or S302N) in the LS did not significantly affect the redox regulation (supplemental Fig. S5). Collectively, these results suggest that the LS is not directly involved in the redox regulation of the potato tuber AGPase.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
The exact role of the LS in the functioning of the heterotetrameric AGPase from potato tuber remains largely unresolved as unlike the SS, which is capable of forming a catalytically active homotetrameric enzyme, the LS is largely insoluble and forms inclusion bodies when expressed as a recombinant form in bacterial cells. Although crude insect cell extracts containing the barley endosperm LS (bepL10) were devoid of AGPase activity (29), bacterial cell extracts expressing the potato (2), or maize endosperm (30) LS displayed AGPase activity at levels slightly higher or comparable to cell extracts containing expressed SS suggesting that the LS possesses catalytic activity. However, in all cases no further efforts were undertaken to purify and enzymatically characterize the small amounts of soluble LSs present in these crude extracts.

Because the homotetrameric LS enzyme form was not available until now, the role of the LS has been indirectly inferred based on the properties of the heterotetrameric enzyme containing SS variants (6, 18, 19, 21, 31, 32). Mutations in conserved residues putatively required for Glc 1-P binding or for catalysis in the SS but not in the LS drastically lowered enzyme activity (18), suggesting that SS was solely responsible for catalysis of the heterotetrameric enzyme. This view was also supported by the restoration of catalysis of the heterotetrameric enzyme containing the catalytically silenced D143N SS mutant (SSi) by the introduction of K41R and/or T51K substitutions in the LS (7). Although these results assume that the observed catalytic activity of this mutant AGPase is generated exclusively by the LS, direct insights on its role in enzyme function are more likely to be gained by isolation and enzymatic characterization of a homotetrameric LS.

In this study, we demonstrate that a single mutation, S302N, significantly increases the solubility of the LS and, thereby, facilitating its self-assembly to form a quaternary structure. This condition enabled us to study the enzymatic properties of the LN homotetramer and compare it to the SS homotetramer and LS-SS heterotetrameric forms. The LN homotetramer is catalytically very inefficient as its enzyme activity (0.4 unit/g) is just above background controls under our assay conditions (Table 1). The extent of LN activity is comparable to the catalytic silenced SSi homotetramer (0.3 unit/g). When assembled together, the resulting LNSSi exhibits enzyme activity (0.4 unit/g) in the absence of 3-PGA comparable to the LN and SSi homotetrameric forms. In the presence of 3-PGA, however, the LN responds differently depending on whether it is in the homotetrameric or heterotetrameric form with SSi. The LN (as well as the LRKN) homotetramer does not respond to 3-PGA but, when assembled with SSi, shows a 30-fold increase in enzyme activity. This enhancement is more readily evident when one compares the activities of LRKN and SSi homotetramers to the heterotetrameric enzyme composed of these subunit types. Enzyme activity of LRKNSSi is 3400 units/g, whereas those for LRKN and SSi homotetramers are 320 units/g and 0.3 unit/g, respectively. The LRKNSSi enzyme displays a specific activity 11-fold greater than that individually contributed by LRKN and SSi homotetramers (Table 1). Hence, the catalytic potential of LRKN is suppressed in the homotetrameric form and/or is activated in the heterotetramer. This behavior is much different from that exhibited by the SWT homotetramer where only the allosteric regulatory properties are affected by the absence of the LS. This enhancement of the catalytic rate by the LN and LRKN subunits when assembled with catalytic-silenced SSi indicates that the LS 3-PGA binding sites are functional when operating in conjunction with the SS but not in the homotetrameric form (Table 1 and Fig. 2). Overall, these results show that the LS possesses catalytic activity, albeit much lower than the SS. Moreover, the increased responsiveness to 3-PGA activation by the heterotetramers (LNSSi and LRKNSSi) compared with homotetramers (LN, LRKN, and SSi) re-confirms our earlier conclusion that the enzymatic properties of AGPase are a product of synergism between LS and SS interactions (6, 9).


Figure 6
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  		FIGURE 6.
Modeled structures of LWT (A) and LN (B).

 
Wild-type AGPase heterotetramer shows hyperbolic activation by 3-PGA (Fig. 2A). The SWT homotetramer also displays a typical hyperbolic activation curve in response to elevating 3-PGA concentrations but its A0.5 value (~104 µM) is >130-fold larger than the wild-type enzyme (Fig. 2, D versus A, and Table 1). Interestingly, the LS homotetramers (LRKN and LN) were unresponsive up to 1 mM 3-PGA, and LRKN showed a slight inhibition in activity at higher 3-PGA concentrations (Fig. 2, E and F). This lack of 3-PGA activation by LRKN is somewhat surprising, because the activator mimic, pyridoxal phosphate, specifically labels two lysine residues at positions 415 and 453 (25), which are conserved in the SS. Moreover, site-directed mutagenesis of these lysine residues in the LS lowered the sensitivity of the heterotetrameric enzyme (3- to 12-fold) to 3-PGA activation (31). Although similar substitutions in these conserved Lys residues in the SS resulted in a much greater (54- to 3090-fold) inactivation, these results support the involvement of the LS in determining the net allosteric regulatory properties of the heterotetrameric enzyme. This view is best supported by the regulatory properties of the LNSSi, LRKSSi, and LRKNSSi enzymes whose catalytic activities are contributed nearly solely by the LS in the presence of 3-PGA (Table 1), because the SSi lacks the essential Asp for binding of the catalytic metal ion. These heterotetrameric enzymes show a 30-fold or more increase in catalytic activity in the presence of 3-PGA.

In addition to its very low catalytic turnover properties, the LRKN homotetramer possesses poor substrate affinities compared with the wild-type SS homotetramer: ~10-fold lower affinities for ATP and 12-fold lower for Glc 1-P irrespectively of whether or not 3-PGA is present. This reduction in ATP affinity by the LRKN homotetramer is also readily evident when probed with the 8-N3-ATP, which exhibits a labeling constant ~3- to 4-fold higher than that seen for the LRKNSWT or SWT homotetramer. This less efficient labeling of LS in the homotetrameric form as compared with more efficient labeling in the heterotetrameric form (6) indicates that ATP binding sites in the LS homotetramer form have an altered conformation than when the LS is assembled with the SS. Nevertheless, the efficient labeling of the LS with 8-N3-ATP (Fig. 4) (6) and the ability to elevate the enzyme activity of the heterotetrameric enzyme containing the catalytically silenced SS or homotetrameric LS by the introduction of K41R and T51K substitutions indicate that the substrate binding sites of the LS have been preserved, but the catalytically capacity of this subunit has been compromised (7).

Kinetic analysis revealed that the LRKNSSi heterotetramer mutant shows a 4-fold increase in 3-PGA sensitivity over that of LRKSSi (Table 1) with only a marginal change in catalytic rate (Table 2). This enhanced up-regulatory properties gained by the S302N mutation is likely the primary basis responsible for the elevated glycogen production by bacterial cells. Interestingly, Asn at position 302 is highly conserved in nearly all other wild-type AGPase sequences: the only exceptions are two tomato LSs (GenBankTM accession nos. U88089 and U81033). To date, three LS forms have been identified from potato (33). It should be noted that the other two LS forms (GenBankTM accession nos. X76136 and X74982) possess an Asn residue at position 302, implicating analogous function with the LN mutant. Our modeled LS structure (Fig. 6) shows that substitution of Ser302 to Asn generates a new hydrogen bond between the side chain of Asn302 and backbone of Gly57 in the glycine-rich loop, which is closely associated with the regulation and catalysis of the enzyme (6, 7, 9, 21, 32). Thus, this change in the loop structure is likely responsible for the up-regulatory properties of the AGPase heterotetramer. Moreover, this conformational change also increases the solubility of the subunit most likely by facilitating folding of the polypeptide to a mature soluble state. However, S302N replacement does not significantly affect the redox status of the enzyme (Fig. 7).


Figure 7
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  		FIGURE 7.
Reduction of AGPase heterotetramers and homotetramers with DTT. Purified AGPase heterotetramers (A–C) and LS homotetramers (E and F) and the partially purified AGPase SS homotetramer (D, purified using DEAE-Sepharose FF and POROS 20HQ purification columns) were boiled for 5 min in a SDS sample buffer containing 62.5 mM Tris-HCl (pH 6.8), 2% w/w SDS, 7.5% v/v glycerol in the absence (–) or presence (+) of 50 mM DTT. 0.2 µgof proteins (2 µg for SWT) was applied to SDS-PAGE and immunoblot analysis. Antibodies used were anti-potato tuber AGPase LS or SS. A, LWT SWT; B, LRKN-SWT; C, LNSWT; D, SWT; E, LRKN; and F, LN.

 
In conclusion, isolation and characterization of the LS homotetramer of the potato tuber AGPase enabled us to demonstrate that the potato tuber LS possesses catalytic and regulatory properties, which are distinct from the SS homotetramer. These properties differ heavily depending on its quaternary form. Other than functional substrate binding and possibly effector binding sites, the LS homotetramer is essentially devoid of allosteric regulatory properties. However, the catalytic potential of LS is partially unmasked, and responses to 3-PGA activation are restored when assembled with the catalytic-silenced D143N SS. Taken together, these results strongly support our idea that the catalytic and allosteric properties of L2S2 are not a simple combination of the properties of the homotetrameric LS (L4) and SS (S4) but a product of subunit synergy. Further structural and biochemical studies of the LRKN, LN, and SWT homotetramers are underway to get more insights on the evolution of the AGPase subunits as it relates to the structure-function relationship.


   FOOTNOTES
 
* This work was supported by Dept. of Energy Grant DE-FG02-96ER20216 and falls under the purview of the Hatch Regional NC-1142 Project. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

Formula The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1–S5 and Tables S1 and S2. Back

1 To whom correspondence should be addressed: Institute of Biological Chemistry, Washington State University, Pullman, WA 99164-6340. Tel.: 509-335-3391; Fax: 509-335-7643; E-mail: okita{at}wsu.edu.

2 The abbreviations used are: AGPase, ADP-glucose pyrophosphorylase; DTT, dithiothreitol; Glc 1-P, glucose 1-phosphate; L or LS, large subunit; 3-PGA, 3-phosphoglycerate; S or SS, small subunit. Back



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 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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