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Plant Physiol. (1998) 118: 265-274
ADP-Glucose Pyrophosphorylase from Potato Tubers.
Site-Directed Mutagenesis Studies of the Regulatory Sites1
Miguel A. Ballicora,
Yingbin Fu,
Natasha M. Nesbitt, and
Jack Preiss*
Department of Biochemistry, Michigan State University, East
Lansing, Michigan 48824
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ABSTRACT |
Several lysines (Lys) were determined
to be involved in the regulation of the ADP-glucose (Glc)
pyrophosphorylase from spinach leaf and the cyanobacterium
Anabaena sp. PCC 7120 (K. Ball, J. Preiss [1994]
J Biol Chem 269: 24706-24711; Y. Charng, A.A. Iglesias, J. Preiss
[1994] J Biol Chem 269: 24107-24113). Site-directed mutagenesis
was used to investigate the relative roles of the conserved Lys in the
heterotetrameric enzyme from potato (Solanum tuberosum
L.) tubers. Mutations to alanine of Lys-404 and Lys-441 on the small
subunit decreased the apparent affinity for the activator, 3-phosphoglycerate, by 3090- and 54-fold, respectively. The apparent affinity for the inhibitor, phosphate, decreased greater than 400-fold.
Mutation of Lys-441 to glutamic acid showed even larger effects. When
Lys-417 and Lys-455 on the large subunit were mutated to alanine, the
phosphate inhibition was not altered and the apparent affinity for the
activator decreased only 9- and 3-fold, respectively. Mutations of
these residues to glutamic acid only decreased the affinity for the
activator 12- and 5-fold, respectively. No significant changes were
observed on other kinetic constants for the substrates ADP-Glc,
pyrophosphate, and Mg2+. These data indicate that Lys-404
and Lys-441 on the small subunit are more important for the regulation
of the ADP-Glc pyrophosphorylase than their homologous residues in the
large subunit.
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INTRODUCTION |
ADP-Glc PPase (ATP: -Glc-1-P adenylyltransferase, EC 2.7.7.27)
catalyzes the synthesis of ADP-Glc from Glc-1-P and ATP, releasing PPi
as a product. This reaction is considered a prime regulatory step in
the synthesis of bacterial glycogen and starch in plants (Preiss, 1984 ,
1988, 1991, 1997a).
Potato (Solanum tuberosum L.) tuber ADP-Glc PPase is
activated by 3-PGA and inhibited by Pi, as are most higher-plant,
algal, and cyanobacterial ADP-Glc PPases (Preiss, 1982, 1988, 1991,
1997a). The enzyme from cyanobacteria is homotetrameric, as are other enzymes from bacteria (Iglesias et al., 1991; Kakefuda et al., 1992).
Conversely, all of the ADP-Glc PPases from higher plants studied so
far, including the potato tuber enzyme, are heterotetramers composed of
two distinct subunits (Okita et al., 1990). Even though there is little
difference in molecular mass, use of the terms "large" for the
51-kD subunit and "small" for the 50-kD subunit has been retained,
because they have homology to the large and small subunits,
respectively.
The amino acid sequences of the small subunit of higher-plant ADP-Glc
PPases are highly conserved, but there is less similarity among the
large subunits (Smith-White and Preiss, 1992). It has been suggested
that the major function of the large subunit is to modify regulatory
properties of the small subunit, which is the subunit primarily
involved in catalysis (Ballicora et al., 1995).
The genes of both subunits of the potato tuber ADP-Glc PPase have been
cloned (Nakata et al., 1991). The cDNAs corresponding to the two
subunits of the potato enzyme have been expressed in an
Escherichia coli strain (AC70R1-504) deficient in ADP-Glc
PPase activity (Iglesias et al., 1993; Ballicora et al., 1995). This recombinant enzyme was composed of two large subunits and two small
subunits to yield a heterotetrameric
(L2S2) native structure, as
shown by N-terminal sequencing (Preiss, 1997b). This expression system
allowed us to study the interaction between subunits and the roles
played by each in heterotetrameric enzymes from higher plants.
The first structural study on the regulatory site of the plant enzymes
was performed on the ADP-Glc PPase purified from spinach leaves. After
chemical modification with pyridoxal 5 -phosphate, protection with
effectors, and sequencing of the peptides, covalently modified Lys
residues involved in allosteric regulation were identified. These
residues were involved in the binding of either the activator, 3-PGA,
or the inhibitor, Pi, (Ball and Preiss, 1994). Three of the Lys
residues are highly conserved in the enzymes from higher plants and
cyanobacteria (Table I, s-II, l-I, l-II).
Two are equivalent (s-II and l-II), one corresponding to the large
subunit and the other to the small subunit (Lys-440 in the small
subunit of spinach leaf and Lys-441 in that of potato tuber). The third Lys residue labeled by pyridoxal 5-phosphate was present in the large
subunit (l-I; Table I). The enzyme from Anabaena, which is
composed of only one type of subunit, conserves these Lys residues in
the primary structure (sites I and II; Table I).
Using chemical modification and site-directed mutagenesis techniques
(Charng et al., 1994; Sheng et al., 1996), both Lys-382 and Lys-419
were shown to be involved in the activation by 3-PGA. However, no
studies were performed to replace these residues on an enzyme
consisting of two different subunits, which would be necessary to
understand the role that each subunit plays in higher-plant enzymes. In
the present study site-directed mutagenesis was used to determine
whether the Lys residues of both subunits are involved in the
activation by 3-PGA or the inhibition by Pi of the potato tuber ADP-Glc
PPase. It was found that the effects of substitution of the Lys
residues in the large and small subunit are not equivalent. Mutating
Lys-404 and Lys-441 on the small subunit decreased the affinity for
the activator 3-PGA and the ability of the enzyme to be inhibited by
Pi. However, the effects were less pronounced when the homologous
mutations were performed on the large subunit.
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MATERIALS AND METHODS |
Reagents
32PPi was purchased from DuPont-New England Nuclear.
[14C]Glc-l-P was from ICN Pharmaceuticals.
[-35S]dATP was from Amersham.
Oligonucleotides were synthesized and purified by the Macromolecular
Facility at Michigan State University (East Lansing). Restriction
enzymes were from New England Biolabs. All other reagents were of the
highest quality available.
Bacterial Strains and Media
Escherichia coli strain TG1 (K12,  [lac-pro], supE,
thi, hsdD5/F'traD36, proA+B+, lacIq, lacZM15) was used for
site-directed mutagenesis and grown in 2× YT medium (Sambrook
et al., 1989). E. coli mutant strain AC70R1-504, which is
deficient in ADP-Glc PPase activity, was used for expression of the
wild-type and mutant enzymes described in this work. For the
preparation of plasmids pMON17336 and pMLaugh10, AC70R1-504 was grown
in Luria-Bertani medium (Sambrook et al., 1989) in the presence
of kanamycin (25 µg/mL) or spectinomycin (70 µg/mL), respectively.
Plasmids and Phages
Plasmid pMLaugh10 is a vector used for the expression of an insert
that encodes the small subunit of ADP-Glc PPase from potato (Solanum tuberosum L.) tubers. pMON17336 is another
expression vector, compatible with pMLaugh10, that contains an insert
that encodes the large subunit. The construction of both plasmids was described previously (Iglesias et al., 1993; Ballicora et al., 1995).
Phages M13 mp18 and M13 mp19, derivatives of M13, were used for the
preparation of single-stranded DNA (Messing, 1983).
Site-Directed Mutagenesis
Site-directed mutagenesis was performed using a previously
published procedure (Sayers et al., 1988) and an in vitro mutagenesis kit (Sculptor, Amersham). Because this method requires a
single-stranded DNA template, target genes were subcloned to vectors
capable of producing it. For that purpose, phages M13 mp18 and M13 mp19
were used as follows: Plasmid pMON17336 was digested with
XbaI, and the fragment that contained the gene of the large
subunit was subcloned onto M13 mp19, whereas pMLaugh10 was digested
with EcoRI and the fragment that encoded the small subunit
was subcloned onto M13 mp18. Single-stranded DNA was prepared from
these phages to proceed with the site-directed mutagenesis (Sayers et
al., 1988). The oligonucleotides used to perform the mutations are shown in Figure 1. Once the mutations
were confirmed by dideoxy sequencing (Sanger et al., 1977), replicative
forms (double-stranded DNA) of the phages were prepared and digested
with the restriction enzyme used to introduce the fragment. Those
fragments were returned to the original vectors, pMON17336 and
pMLaugh10, respectively. To verify that there were no unintended
mutations, the entire coding regions of these mutated plasmids were
sequenced. The plasmids with the desired mutations were used to
transform AC70R1-504 cells for expression.
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| Figure 1.
Synthetic oligonucleotides used for site-directed
mutagenesis. Underlined nucleotides denote differences from the
wild-type sequence. Asterisks indicate strands produced from the phage
(M13 mp18 and M13 mp19 for the small and large subunit genes,
respectively) and used as templates on the mutagenesis protocol.
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Expression and Purification of Wild-Type and Mutant Enzymes
Wild-type and mutant genes were expressed in AC70R1-504 cells to
obtain heterotetrameric or homotetrameric enzymes, as described previously (Ballicora et al., 1995) with only one modification. The
concentration of isopropyl- -D-thiogalactopyranoside was
increased from 0.01 to 0.5 mM in the induction. To express
the mutants, cells were transformed with the mutated plasmids pMON17336
or pMLaugh10.
The enzymes were purified as indicated previously with the following
modifications. After the heat treatment step, a precipitation step was
introduced in which ammonium sulfate was added to the extract until
30% saturation was reached. After centrifugation for 15 min at
15,000g, the supernatant was taken to 60% saturation and
centrifuged for 20 min at 15,000g. This pellet was dissolved in a minimal amount of buffer A (Ballicora et al., 1995) and desalted against the same buffer using Econo-Pac 10DG columns (Bio-Rad). All of
these steps were performed at 4°C. Another modification was at the
hydrophobic chromatography step, in which ammonium sulfate was used
instead of potassium phosphate buffer. Other steps remained unchanged
and, unless indicated otherwise, all mutant enzymes reached >90%
purity.
Assay of ADP-Glc PPase
Assay A: Pyrophosphorolysis Direction
Pyrophosphorolysis of ADP-Glc was determined by the formation of
[32P]ATP from 32PPi.
Unless indicated otherwise, the reaction mixture contained 20 µmol of
Gly-Gly buffer, pH 8.0, 1.75 µmol of MgCl2,
0.75 µmol of DTT, 2.5 µmol of NaF, 0.5 µmol of ADP-Glc, 0.38 µmol of 32PPi (0.5 × 106 cpm µmol 1 to
2.0 × 106 cpm
µmol1), 50 µg of crystalline BSA, and
variable concentrations of 3-PGA in a final volume of 0.25 mL. The
reaction was started by the addition of enzyme and after a 10-min
incubation at 37°C was terminated by the addition of 3 mL of cold 5%
TCA. The [32P]ATP formed was measured as
described previously (Morell et al., 1987). Inhibition by Pi was
measured by the addition of potassium phosphate, pH 8.0, to the
reaction mixture.
Assay B: Synthesis Direction
Synthesis of ADP-Glc was followed by the formation of
[14C]ADP-Glc from
[14C]Glc-1-P. Reaction mixtures contained (in
0.2 mL): 20 µmol of Hepes buffer, pH 8.0, 1 µmol of
MgCl2, 0.6 µmol of DTT, 0.1 µmol of
[14C]Glc-1-P (1.0 × 106 cpm µmol1), 0.3 µmol of ATP, 0.3 unit of inorganic pyrophosphatase, and 40 µg of
crystalline BSA. 3-PGA was added at different concentrations as
indicated. Assays were initiated by addition of the enzyme. Reaction
mixtures were incubated for 10 min at 37°C and terminated by heating
in a boiling-water bath for 1 min. [14C]ADP-Glc
was assayed as described previously (Ghosh and Preiss, 1966). In both
assays A and B, 1 unit is defined as the amount of enzyme that produces
1 µmol of product in 1 min. For assay of the mutant enzymes, the
reaction conditions were identical to the wild type except that
the amount of 3-PGA was altered to obtain maximal activity.
Protein Assay
Protein concentration was determined using bicinchoninic acid
reagent (Smith et al., 1985) (Pierce) with BSA as the standard.
Kinetic Characterization
Kinetic data were plotted as velocity versus substrate or effector
concentration. Kinetic constants were obtained from Hill plots and were
confirmed through nonlinear least-squares fitting of the data with the
Hill's equation or the Michaelis-Menten equation for hyperbolic plots
(Fraser and Suzuki, 1973).
Kinetic constants were expressed as A0.5,
S0.5, and I0.5,
which correspond to the concentration of effector necessary to reach 50% of maximal activation, velocity, and inhibition, respectively. The
-fold activation was the ratio between the maximal activity obtained at
saturating concentrations of activator and the activity in the absence
of it.
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RESULTS |
Effect of the Mutations on the Pyrophosphorolysis Direction
Using partially purified enzymes, it has been reported that in the
presence of the large subunit from potato tubers, the small subunit has
a higher affinity for the activator 3-PGA in both the synthesis and
pyrophosphorolysis directions (Ballicora et al., 1995). In the present
study using proteins purified to homogeneity, we observed that the
homotetramer composed of only the small subunit (S4) and
the heterotetramer (L2S2)
had similar specific activities at saturated concentrations of 3-PGA in
the pyrophosphorolysis reaction. They were 55 and 48 units
mg1, and the A0.5
for 3-PGA was 900 and 2.2 µM, respectively (Table II). Figure
2 shows representative activation curves
for the wild-type and
LK417ASwt ADP-Glc PPases.
In addition to the affinity for the activator, another difference was
noted: When the activity was analyzed in the absence of 3-PGA, the
activity of the small subunit alone was negligible; when it was
expressed in the presence of the large subunit, the activity in the
absence of activator was 13.3 units mg1, which
is only 3.6-fold lower than the maximal activity at saturated concentrations of 3-PGA (Table II). Because the presence of the large
subunit in the quaternary structure is so important in allosteric activation, it was of interest to determine the effect of mutations on
the putative allosteric sites of that subunit.
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Table II.
Activation by 3-PGA in the pyrophosphorolysis
reaction
E. coli AC70R1-504 cells were cotransformed with
two plasmids. Plasmid pMON17336 encodes either the wild-type (wt) large
subunit of the potato tuber ADP-Glc PPase or a mutated large subunit.
The other plasmid (pMLaugh10) encodes either the wild-type small
subunit or a single amino acid mutant. Enzymes were expressed and
purified as described in ``Materials and Methods''. Specific
activities of the mutants were determined at saturating concentrations
of activator (3-PGA) and substrates. Every constant was determined at
least twice and the difference was <10% of the average in all cases.
Average values are shown.
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| Figure 2.
The effect of 3-PGA concentration on ADP-Glc PPase
activity in the pyrophosphorolysis direction. For the wild-type
enzyme and the mutants LK417ASwt and
LwtSK404A, 100% activity represents
42, 12, and 5.2 µmol of ATP produced min1
mg1, respectively.
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When the Lys-455 on the large subunit was mutated, the apparent
affinity for 3-PGA decreased. The A0.5
values were 3- and 5-fold higher when the residue was replaced by Ala
and Glu, respectively. Greater changes were observed when large-subunit
Lys-417 was mutated. In this case, the A0.5
values were 9- and 12-fold higher when the residue was replaced by Ala
or Glu, respectively (Table II). The catalytic ability of the enzyme
was not affected dramatically by these four mutations. However, a
greater reduction was observed when the activity was assayed in the
absence of 3-PGA. For that reason, the activation increased from 3.6 (wild type) to 6, 22, 18, and 50 for the mutations K455A, K417A, K417E,
and K455E, respectively (Table II). Even though these mutated large
subunits produced heterotetrameric enzymes with lower affinity for the
activator than the heterotetrameric wild type, the mutated large
subunits still had the ability to increase the apparent affinity of
3-PGA for the small subunit when both subunits were expressed together.
When the wild-type small subunit was expressed along with the mutated
large subunits K455A, K455E, K417A, and K417E, the
A0.5 for 3-PGA was reduced from 900 µM to 6, 10, 20, and 27 µM, respectively (Table II).
Mutations of the homologous residues on the small subunit were
performed to determine whether they are more relevant than their
equivalent residues on the large subunit to the activation of the
enzyme. When Lys-441 was replaced with Ala or Glu, the A0.5 for 3-PGA increased 54- and 191-fold,
respectively. However, the specific activities at saturated
concentrations of activator were not much different than those of the
wild type: 32 units mg1 for the Ala and 39 units mg1 for the Glu mutant. The activity of
these mutants in the absence of 3-PGA did not change much. For that
reason, the maximal activation remained at 2.9 for the Ala and 3.1 for
the Glu mutant (Table II). The greatest decrease in apparent affinity
was obtained when Lys-404 on the small subunit was replaced by Ala. The
A0.5 for 3-PGA was 6800 µM,
which represents a 3090-fold increase over the wild type (Table II;
Fig. 2). Despite this mutant being severely affected in its allosteric
properties, the maximal specific activity was 12 units
mg1, only 4 times lower than the wild type.
Conversely, the pyrophosphorolysis activity was very dependent on the
allosteric activator, as shown by the fact that 3-PGA activated the
K404A-mutated enzyme 130-fold (Table II).
Two double mutants were prepared by cotransforming with plasmids that
encoded subunits with a single mutation. These double mutants had a
remarkable effect on the activation properties. Not only were the
A0.5 values for 3-PGA of the
LK417ASK441A and LK417ESK441E enzymes much
higher than that of the wild type, but they were also higher than the
A0.5 of each single mutant. Moreover, the
effects of these mutations seemed to be additive. The
A0.5 for 3-PGA of
LK417ASK441A was 177-fold
higher than that of the wild type, which is close to the value that
would be expected (162-fold) if the LK417A
mutation increased 3-fold and the SK441A mutation
contributed with a 54-fold increase, as occurred when each single
mutant was analyzed (Table II). At the same time, the
A0.5 for 3-PGA of
LK417ESK441E was 2955 times
higher than that of the wild type. If each mutation contributed to the
same extent that they did in the single mutants (5- and 191-fold
increase of the A0.5 for 3-PGA), an
increase of 955-fold would be expected. The effect observed with the
double mutant was higher but in the same range.
There was a common property between the double and the single mutants
on the LK417 site that has not been observed with
the mutations on the SK441 residue. Because the
activities in the absence of 3-PGA decreased significantly, the
activation by 3-PGA was very high for both the
LK417ASK441A and
LK417ESK441E mutants: 80- and 96-fold, respectively (Table II). A role in catalytic efficiency
can be ruled out for these residues (LK417 and
SK441). The specific activities of the double
mutants were lower than those of the wild type; however, this decrease
is not significant compared with the effect on the apparent affinity
for the activator (Table II).
Effect of the Mutations on the Inhibition by Pi
It has been shown for spinach leaf ADP-Glc PPase that the
activation and the inhibition are structurally related. Two Lys residues were protected by both 3-PGA and Pi against chemical modification by pyridoxal 5-phosphate (Ball and Preiss 1994). In
the present study the mutations performed on the ADP-Glc PPase from
potato tubers yielded enzyme forms with altered activation properties.
Therefore, it was of interest to determine whether the inhibition by Pi
was also altered. For this purpose, the mutants were analyzed in the
pyrophosphorolysis direction because the activities were measurable
even in the absence of the activator 3-PGA. Furthermore, in this
direction all of the mutants were activated by 3-PGA, so inhibition
studies could be performed not only in the presence but also in the
absence of the activator.
To measure inhibition in the absence of activator, the activity was
analyzed in the absence of 3-PGA at different concentrations of Pi for
determination of the I0.5. None of the
mutations performed in the large subunit decreased the affinity for the
inhibitor Pi. On the contrary, those mutants were equally or more
sensitive to Pi inhibition than the wild type. The
I0.5 for the wild type was 74 µM, whereas the I0.5 of the
mutants K455A, K455E, K417A, and K417E was 19, 20, 76, and 22 µM, respectively (Table
III). Conversely, when Lys-441 and
Lys-404 on the small subunit were replaced, mutants became virtually
insensitive to Pi inhibition. The I0.5 of
all mutants, K441A, K441E, and K404A, was more than 200-fold higher
than that of the wild type (Table III). These results suggest that the
Lys-441 and Lys-404 residues of the small subunit are important for
inhibition by Pi, whereas Lys-417 and Lys-455 of the large subunit play
little or no role. To confirm this, the effect of Pi in presence of the
activator 3-PGA was also studied.
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Table III.
Inhibition of the ADP-Glc PPase mutants by
phosphate in the pyrophosphorolysis direction
E. coli AC70R1-504 cells were cotransformed with two
plasmids. Plasmid pMON17336 encodes either the wild-type (wt) large
subunit of the potato tuber ADP-Glc PPase or a mutated large subunit.
The other plasmid (pMLaugh10) encodes either the wild-type small
subunit or a single amino acid mutant. Enzymes were expressed and
purified as described in ``Materials and Methods''. The substrates
and cofactors were 2 mM ADPGlc, 1.5 mM PPi, and
7 mM Mg2+. The I0.5 of
Pi was determined as described in ``Materials and Methods''. Every
constant was determined at least twice and the difference was <10% of
the average in all cases. Average values are shown. The 3-PGA
activation curve was performed in presence of 0.5 mM
phosphate.
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Pi can reverse the activation caused by 3-PGA and vice versa.
Therefore, Pi in the reaction mixture shifts the 3-PGA activation curve
toward higher concentrations, thus increasing the
A0.5. To study this effect, an activation
curve was performed for each mutant in the presence of 0.5 mM Pi. The resultant A0.5 was
then compared with that determined in the absence of inhibitor (Table III). When the wild-type enzyme was analyzed, 0.5 mM Pi
increased the A0.5 for 3-PGA 25-fold. All
of the mutations performed in the large subunit produced enzymes that
showed similar results. Pi increased the
A0.5 for 3-PGA by 27-, 28-, 20-, and
22-fold for the mutants K455A, K455E, K417A, and K417E, respectively. Conversely, none of the mutations performed on the small subunit, K441A, K441E, and K404A, exhibited the same effect. Activation curves
in the presence or absence of Pi were very similar. The A0.5 values were little affected,
increasing only 2-, 1.9-, and 1.2-fold for the mutants K441A, K441E,
and K404A, respectively (Table III).
Effect of the Mutations on the Kinetic Constants and Stability
The allosteric properties were altered when Lys-417, Lys-455
(large subunit), Lys-404, and Lys-441 (small subunit) were replaced by
either Ala or Glu. To confirm that these residues were specifically involved in the regulation of the enzyme, kinetic characteristics of
the substrates were determined to see if they were altered. The
affinities for Mg2+ and PPi did not change
dramatically in any of the mutants tested. The
S0.5 for Mg2+
remained between 2.6 and 4.2 mM, and the
Km for PPi did not increase more than
2-fold for any of the mutants relative to the wild type (Table
IV). The apparent affinity for ADP-Glc
did not change for most of the mutants. Double-mutant
LK417ESK441E and
single-mutant LwtSK404A had
Km values of 0.60 and 0.90 mM,
respectively, for ADP-Glc (Table IV). These parameters were 2.7- and
4.1-fold higher than those of the wild type. However, these changes are
small compared with the striking changes in allosteric properties
observed for these two mutants (Tables II and III).
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Table IV.
Pyrophosphorolysis of ADP-Glc: kinetic constants of
substrates and cofactors
E. coli AC70R1-504 cells were cotransformed with two
plasmids. Plasmid pMON17336 encodes either the wild-type (wt) large
subunit of the potato tuber ADP-Glc PPase or a mutated large subunit.
The other plasmid (pMLaugh10) encodes either the wild-type small
subunit or a single amino acid mutant. Enzymes were expressed and
purified as described in ``Materials and Methods''. The concentration
of 3-PGA used was 10 mM. In the case of mutants
LwtSK404A and
LK417ES441E the concentration was 20 mM. The concentration of ADP-Glc, PPi, and Mg2+
were as described in ``Materials and Methods''. Every constant was
determined at least twice and the difference was <10% of the average
in all cases. Average values are shown.
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These results suggest that the mutations had little if any effect on
the catalytic site and that they specifically altered the regulatory
properties. Furthermore, like the wild type, all of these mutants were
stable (>70% recovery) to a treatment at 60°C for 5 min (data not
shown). This was used as a first purification step in all cases and
indicates that the Lys residues under study are not necessary for the
stability of the enzyme structure.
Effect of the Mutations on the Synthesis Direction
The large subunit has the ability to reduce the
A0.5 for 3-PGA when expressed along with
the small subunit. That effect is more marked in the pyrophosphorolysis
direction, so changes in the affinity for the activator were easier to
detect. However, the activation in the reaction of synthesis was also
studied because this is the direction of the reaction in vivo in the
pathway of the starch biosynthesis.
When Lys-455 on the large subunit was mutated to Ala or Glu, the
A0.5 for 3-PGA increased 2- and 8-fold,
respectively. Greater increases in A0.5
were obtained by mutating Lys-417 on the large subunit. The
A0.5 of mutants K417A and K417E were 3 and
13 times higher than that of the wild type (Table
V), indicating that replacing these
residues affected the activation of the enzyme. However, all of these
mutants on the large subunit had an A0.5 for 3-PGA lower than the A0.5 of the small
subunit expressed alone (Table V). As it was observed in the
pyrophosphorolysis direction, mutations on the small subunit caused a
bigger decrease in the apparent affinity for the activator. Mutants
K441A and K441E had A0.5 values for 3-PGA
32 and 83 times higher, respectively, than that of the wild type. At
the same time, mutant K404 could not be activated by 3-PGA, even by
increasing the concentration up to 50 mM.
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Table V.
Activation by 3-PGA of the synthesis of ADP-Glc
E. coli AC70R1-504 cells were cotransformed with two
plasmids. Plasmid pMON17336 encodes either the wild-type (wt) large
subunit of the potato tuber ADP-Glc PPase or a mutated large subunit.
The other plasmids (pMLaugh10) encodes either the wild-type small
subunit or a single amino acid mutant. Enzymes were expressed and
purified as described in ``Materials and Methods''. Specific
activities of the mutants were determined at saturated concentrations
of activator (3-PGA) and substrates (1.5 mM ATP, 0.5 mM Glc-1-P, 7 mM Mg2+). In the case
of mutants LwtSK404A and
LK417ES441E, concentrations of substrates were
increased four times but still no activation was observed. Every
constant was determined at least twice and the difference was <10% of
the average in all cases. Average values are shown. Inhibition has been
observed at higher concentrations of 3-PGA.
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Double mutants also showed that the effects of the mutations were at
least additive. The A0.5 for 3-PGA of
LK417ASK441A was 6.0 mM, which is 60 times higher than that of the wild type.
This is in the range of what is expected if one mutation contributes with an increase of 3-fold and another of 32-fold, as they do when the
single mutants are analyzed. The A0.5 of
LK417ESK441E could not be
measured because the enzyme was not activated by 3-PGA, even up to 50 mM. At the highest concentrations assayed, 3-PGA partially
inhibited LK417ESK441E
(data not shown). In this double mutant, if both Glu mutations
contribute similarly, as they do in the single mutants (with increases
of 13- and 83-fold; Table V), an A0.5 of
approximately 100 mM would have been expected. At such high
concentrations, it is possible that 3-PGA also interacts nonspecifically with other sites of the enzyme, causing inhibition.
Characterization of the LK417MSwt Mutant
The residue Lys-417 in the potato tuber ADP-Glc PPase is highly
conserved among the large subunits from plant enzymes. However, there
are some exceptions (Table I). Because this residue could be involved
in the regulation of the enzyme, a question arose about how relevant a
change would be in the allosteric properties. As has been shown on the
mutant LK417ASwt at this
position, a change from a positively charged to a neutral amino acid
showed a significant but small effect on the activation by 3-PGA and no
effect on the inhibition by Pi. Therefore, some substitutions at this
position could still give an enzyme with the ability to be regulated
allosterically in vivo. To test this postulate, since some enzymes have
a Met at this position (Table I), Lys-417 of the large subunit from
potato tubers was mutated to Met and the regulatory properties were
analyzed (Table VI).
LK417MSwt was activated by
3-PGA in the pyrophosphorolysis and synthesis directions in a manner
similar to that of the Ala mutant.
View this table:
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Table VI.
Regulatory properties of the mutant
LK417MSwt
The mutant LK417MSwt was expressed, purified,
and the kinetic parameters determined as described in ``Materials and Methods''. The substrates and cofactors were 2 mM ADP-Glc,
1.5 mM PPi, and 7 mM Mg2+ in the
pyrophosphorolysis direction, and 1.5 mM ATP, 0.5 mM Glc-1-P, and 7 mM Mg2+ in the
synthesis direction. The results are the average of two independent
experiments and the difference between them was less than 10% in all
cases.
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In the synthesis direction the A0.5 was 450 µM, and in the pyrophosphorolysis direction it was 7 µM; these values are only 4.5 and 3.2 times higher,
respectively, than those of the wild type. At the same time, specific
activities did not change dramatically. In the pyrophosphorolysis
direction it was 45 units mg1, and in the
synthesis direction it was 12 units mg1. The
inhibition by Pi was not altered significantly. The sensitivity toward
Pi in the absence of activator increased from 74 to 20 µM, but the ability to shift the 3-PGA activation curve
remained the same. In the presence of 0.5 mM Pi the
A0.5 for 3-PGA in the pyrophosphorolysis
direction increased 28-fold. Under the same conditions, the wild-type
enzyme showed an increase of 25-fold. The most altered characteristic
between LK417MSwt and the
wild type was that the activation in the pyrophosphorolysis direction increased from 3.6 to 14 because the enzyme was lower in activity in
the absence of the activator. This alteration may have very little
physiological effect.
| |
DISCUSSION |
The allosteric regulation of the ADP-Glc PPase has always been an
important topic because this enzyme catalyzes a key step in the pathway
of the synthesis of glycogen in bacteria and starch in plants (Preiss
1984, 1988, 1991, 1997a, 1997b). To study the different roles of these
sites on a heterotetrameric enzyme, in the current study site-directed
mutagenesis was performed on the homologous Lys residues of the potato
tuber ADP-Glc PPase, which has been cloned and expressed in E. coli cells.
Lys residues (Lys-417 and Lys-455 of the large subunit and Lys-441 of
the small subunit) were mutated conservatively to Arg. However, very
little change was observed on the affinity for the activator. The
A0.5 for 3-PGA in the synthesis direction
did not increase more than 2-fold (data not shown). Therefore, to study the different roles that these residues may play in the large and small
subunits, Ala (neutral) and Glu (negatively charged) mutations were
performed. The mutant enzymes obtained were expressed, purified, and
characterized kinetically. Much greater effects on the affinity for
activator were found when the Lys residues were replaced by Ala and
Glu. The A0.5 for 3-PGA of these mutants increased up to 8.3 mM (83-fold higher than in the wild
type) when Lys-441 on the small subunit was mutated to Glu (Table V). This indicated that the most important characteristic of these Lys
residues is the positive charge that probably interacts with the
negative charge of the phosphate and/or carboxyl groups of the 3-PGA.
At the same time, replacing the Lys-404 on the small subunit
(homologous to Lys-417 of the large subunit) with Ala was enough to
make this mutant insensitive to the activation in the synthesis direction. When the pyrophosphorolysis reaction was analyzed, this
mutant had an A0.5 that was 3090-fold
higher than that of the wild type. The other mutants also had lower
affinities for 3-PGA, but even so, the kinetic constants for the
substrates ADP-Glc, PPi, and the cofactor Mg2+
did not change significantly (Tables II and IV). The most significant difference between these mutants was that mutations on the small subunit showed a much lower affinity for 3-PGA than the mutations on
the large subunit (Tables II and V). Thus, both of these sites in the
small subunit appear to be more important for regulation than the
homologous Lys residues in the large subunit.
It has been thought that the large subunit plays the most important
role in the regulation because its presence in the heterotetramer increases the affinity for the activator 3-PGA (Ballicora et al., 1995). One of the possibilities that was considered was that the large
subunit provides a regulatory site with higher affinity. However,
performing mutations in the large subunit of the putative amino acids
involved in the binding of the activator did not support this idea.
These large-subunit mutants still increased the affinity for 3-PGA when
they were combined with the wild-type small subunit. This effect was
observed whether the reaction was in the synthesis or the
pyrophosphorolysis direction (Tables II and V). Thus, the large subunit
of the potato tuber ADP-Glc PPase increases the affinity for the
activator, but the integrity of its own regulatory-site residues is not
necessary to see this effect.
Furthermore, mutations on the homologous sites on the small subunit
showed larger effects on the regulation of the enzyme. All of this
suggests that the role of the large subunit is to modulate the
regulatory properties of the small subunit rather than to provide a
more efficient allosteric site for activation. The small subunit has
not only a functional catalytic site but also a functional yet
relatively inefficient regulatory site (with a low affinity for the
activator). In the presence of the large subunit, this site in the
small subunit is more efficient as an activator.
The inhibition by Pi showed a very clear difference between the
residues on the small and large subunits. When Lys-441 and Lys-404 on
the small subunit were mutated to either Ala or Glu, the inhibition by
Pi in the absence of the activator almost disappeared. The
I0.5 increased more than 2 orders of
magnitude (Table III). These mutants also lost the ability to shift the
activation curve by the presence of inhibitor. Thus, Pi cannot
effectively compete with the activator. Nevertheless, when the
mutations were performed on the large subunit the effect was very
different. None of the mutants on the Lys-417 or the Lys-455 could
increase the I0.5 for Pi in absence of
3-PGA. In these mutants Pi shifted the activation curve to the same
extent as in the wild type (Table III). Therefore, Lys-404 (site s-I)
and Lys-441 (site s-II) are important for inhibition by Pi. The
homologous sites on the large subunit do not seem to have the same
role. It is interesting that in the spinach leaf enzyme Pi can prevent
the binding of pyridoxal 5-P to the large subunit site (l-I) (Ball and
Preiss, 1994). This can be explained by Pi binding and partially
blocking the access of the activator or chemical analog (pyridoxal 5-P)
to that residue. Recently, another Pi-binding site was found on the
enzyme from Anabaena (Sheng and Preiss, 1997). Because this
enzyme is a homotetramer, it would be interesting to know if this
residue plays a different role in the large or small subunit of the
heterotetrameric higher-plant enzyme.
The results obtained in the present study strongly suggest that the
different subunits have an asymmetric regulatory function in the
heterotetrameric ADP-Glc PPase. Previously, it has been suggested that
they have different roles in catalysis. The small subunit seemed to
have all of the catalytic activity of the heterotetramer, because the
large subunit expressed alone did not show ADP-Glc PPase activity
(Iglesias et al., 1993; Ballicora et al., 1995). Site-directed
mutagenesis experiments on the Glc-1-P site confirmed the idea that the
role of the large subunit on catalysis is minimal (Fu et al., 1998). We
propose that the main role of the large subunit is to interact with the
small subunit and modulate the properties of the activator site. The
structure of the putative activator sites in the large subunit would
not be as important as the overall interaction of the large subunit
that allows the small subunit to increase its affinity for the
activator. This effect could be the result of an induced conformational
change or a direct interaction between regulatory sites. This
hypothesis explains why the Lys residues identified as part of the
activator site showed a higher conservation in the small subunit than
in the large subunit of the plant enzymes sequenced so far. The only time that the Lys residue of the site s-I was not conserved was in a
clone from sugar beet that had a deletion of 12 amino acids in that
region. However, this clone has not been expressed to determine if it
has any activity and it was used only as a probe in blot experiments
(Muller-Röber et al., 1995). Another exception is a PCR product
from Arabidopsis. However, the alteration occurs in the region where
degenerate primers were used (Villand et al., 1993). Another
clone of the small subunit of the enzyme from Arabidopsis leaves has been isolated and the Lys at site s-I is
conserved (B. Smith-White, unpublished results).
It has been suggested that Lys-417 (site l-I, Table I) in the large
subunit is not conserved and may provide different grades of activation
for 3-PGA to different ADP-Glc PPases from different sources (Martin
and Smith, 1995). However, a change in this residue can explain only
slight decreases in affinity for the 3-PGA. Mutating Lys-417 to Met, a
residue that is present in this position in some enzymes, showed little
change in the regulatory properties (Table V). Thus, enzymes bearing a
Met instead of a Lys at this position can still have all of the
properties needed to regulate the synthesis of starch in vivo. If an
ADP-Glc PPase from a higher plant does not show the classic regulation
by 3-PGA and Pi, it is not because there is a Met in this site, so
other structural reasons have to be found.
We have shown that the Lys residues on the small subunit were more
important than the respective homologous residues in the large subunit.
Still, some variations could exist in the relative importance of these
two sites in the small subunit. In the potato tuber ADP-Glc PPase, site
s-I (Lys-404 of the small subunit) seems to be more important than site
s-II (Lys-441 of the small subunit). In other enzymes the opposite
effect cannot be discounted. For instance, in the spinach leaf enzyme,
site s-I is not labeled by pyridoxal 5-P (Ball and Preiss, 1994). This
could be attributable to s-II being more important for regulation than
s-I or because pyridoxal 5-P does not bind for steric reasons. For
instance, when Lys-419 (homologous to s-II and l-II) in the enzyme from Anabaena was mutated, Lys-382 (homologous to s-I and l-I)
became the preferred site for labeling (Charng et al., 1994).
| |
FOOTNOTES |
1
This work was supported in part by Department of
Energy grant no. DE-FG02-93ER20121.
*
Corresponding author; e-mail preiss{at}pilot.msu.edu; fax
1-517-353-9334.
Received March 30, 1998;
accepted May 28, 1998.
| |
ABBREVIATIONS |
Abbreviations:
3-PGA, 3-phosphoglycerate.
PPase, pyrophosphorylase.
| |
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Top
Abstract
Introduction