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Activity Assays ADPG Synthesis Direct Assay This assay is used to measure the rate of conversion of glucose-1-phosphate (G1P) and ATP to ADP-glucose (ADPG) and inorganic pyrophosphate (PPi). The substrate G1P, labeled with 14C, is converted to [14C] ADPG PPase which is subsequently separated from excess substrate via binding to positively charged DE-81 filter discs and quantified via liquid scintillation counting (LSC). Post assay, the left over G1P is digested by alkaline phosphatase allowing for the uncharged labeled glucose to be washed from the filter. This assay is used to determine physiologically-relevant kinetics of the purified enzyme. Pyrophosphorolysis Assay This assay allows for the measurement of activity in the physiologically “reverse direction” conversion of ADPG and PPi to G1P and ATP. The formation of product, [32P] ATP, was measured by quantifying the amount of [32P] ATP (with 32P phosphate released via acid hydrolysis) after separation by binding to activated charcoal using LSC. Under our conditions, the assay is specific and accurate during purification and avoids the potential for randomization of the label if the synthesis assay was used.
Starch is a complex carbohydrate essential to plants and bacteria as an energy storage compound.
The rate-limiting step in starch biosynthesis is catalyzed by the allosteric enzyme ADPGlucose
pyrophosphorylase (ADPG PPase) thereby making it an attractive candidate for protein engineering to
produce a higher yield of of the biodegradable and renewable carbon source starch. In Agrobacterium
tumefaciens (Ag.t), ADPG PPase is activated by fructose-6-phosphate (F6P) and pyruvate and is
inhibited by phosphate and sulfate. Previous molecular models of ADPG PPase have indicated that the
enzyme consists of a homotetramer structure composed of an N and C terminal connected by a loop
region. This loop region, including P288, had been determined to be important in allosteric regulation of
the E. coli enzyme. In addition, x-ray crystal structure of the Ag.t enzyme indicates that E304 is part of
an allosteric site that is comprised of residues from both the N and C terminus. In order to test these
hypotheses, the P288D and E304A and E304D enzymes were generated by site-directed mutagenesis
and expressed and purified for kinetic and physical studies. Confirming previous work, the P288D
enzyme was shown to exhibit high activity in the absence of activator and was relatively insensitive to
both activators and inhibitors. Large scale pure preparations of P288D have been prepared via phenyl
sepharose, anion exchange, and Cibacron Blue chromatography for future crystallization trials. A total of
2.4 mg (1.63 mg/mL) of pure P288D enzyme was obtained. Activator saturation plots were performed
for the E304A and E304D enzymes; the A0.5 values for fructose-6-phosphate (F6P) and pyruvate were
found to be 8.1 µM (fold activation 4.18) and 10 µM (4.24 fold activation), respectively, for the E304A
enzyme and 43.5 µM (fold-activation 3.69) and 170.2 µM (fold-activation 2.66), respectively, for the
E304D enzyme. In addition, the E304D enzyme was activated by FBP (A0.5 = 0.40 mM (fold-activation
2.34). The wild-type enzyme displays A0.5 values ~10 fold higher than the E304A enzyme and fold
activation for F6P and pyruvate 3 and 2 fold higher than E304A, respectively. The A0.5 values for E304D
were more similar to wild-type while the fold-activation for F6P and pyruvate were ~6-fold and ~3-fold
lower than wild-type. The results indicate that both the long loop structure and the site that includes
E304 are important for regulation. Grant number is P031C110116.
Small scale or large scale growth and
induction in EA345 E. coli cells
Cell and Protein Harvesting
ADPG PPase Purification
Kinetic Characterization
Emmanuel Silva1, Hoomai Karzai1, Vonice Benjamin1 ,Andrew Orry2, and C. R. Meyer1 1Chemistry and Biochemistry Department at California State University, Fullerton and 2Molsoft LCC, La Jolla, CA
Uno Q6
(Anion Exchange Column)
Phenyl Sepharose
(Hydrophobic Column)
(NH4)2SO4 Precipitation
(30% Saturation)
Heat Step
(5 minutes at 65˚C)
Blue A Column
(Affinity Column)
Organism Activators Inhibitors .
E. coli FBP AMP, ADP
Rs. r. Pyruvate None
Ag. t. F6P, Pyruvate Pi, ADP, AMP, Sulfate
Rb. s. F6P, FBP, Pyruvate AMP, ADP, Pi, PEP
T. ma. FBP -
Plants 3PGA Pi
Regulation of ADPG PPase
Table 1. Allosteric regulators of ADPG PPase vary by
organism.
• Starch (in plants and algae) and glycogen (in bacteria) are sources of renewable carbon and energy
• Improving starch synthesis efficiency is important for various industries:
• Food - complex carbohydrate in plant crops (ex. rice, wheat and potatoes)
• Pharmaceuticals, cosmetics, packaging, plastics, and bioethanol – complex and natural starch
• Studying the enzymes critical in starch biosynthesis will allow for protein engineering to
develop specialty starches and to improve starch production efficiency
• ADP-Glucose pyrophosphorylase (ADPG PPase):
• Produced from glgC gene
• Converts glucose-1-P (G1P) and adenosine triphosphate (ATP) to ADP-glucose and
pyrophosphate (PPi)
• Catalyzes the rate-limiting step of the three-step starch biosynthesis pathway (M. A. Ballicora et
al., 2004) (Fig. 1)
• An attractive target for protein engineering to increase the production of renewable carbon
• Regulated differently in different organisms (Fig. 2)
Figure 2. Ag. t. ADPG PPase structure and allosteric site molecular modeling (Cupp-Vickery et al., 2008) depicting (a) a ribbon diagram of
enzyme with space-filling models of sulfate, ATP, G-1-P, (b) the sulfate-binding site, (c) a space-filling model of enzyme with F-6-P (yellow) in
proposed activator-binding site, ATP (orange) and G-1-P (purple) in active site, and basic (blue), acidic (red), and polar (green) residues, and (d) the
proposed F-6-P binding site and possible participating residues. (S: yellow, O: red, C: green, N:blue, P: brown)
ADPG PPase Structure
Starch Biosynthesis Pathway
Glucose-1-phosphate ADP glucose
ADP glucose (glucosyl)4
(glucosyl)5
starch synthase
ADP-glucose pyrophosphorylase
branching enzyme
Figure 1. ADP-glucose pyrophosphorylase (ADPG
PPase) catalyzes the rate-limiting step of starch
biosynthesis in plants and glycogen biosynthesis in
bacteria. ADPG PPase converts glucose-1-phosphate
(G1P) and ATP to ADP- glucose and PPi (upper left).
The activated form of glucose is then added to a
growing glycan chain by starch synthase by a α 1,4
linkage (lower left). The glycan macromolecule
becomes more tightly packed as branching enzymes
reattaches fragments of long, growing glucosyl chains
as branches of other chains (α 1,6 linkages, below).
S0.5(mM) Vmax (U/mg)
ATP Hill #
No Effector 0.21 ±0.04 1.3 ± 0.2 12.1 ±1.0
WT + 2 mM F6P 0.051 ±0.004 1.3 ±0.1 147.9 ±4.1
+ 2 mM Pyruvate 0.10 ±0.01 1.9 ±0.03 105 ±5
No Effector 0.55 ± 0.03 2.9 ± 0.3 52.2 ± 1.9
P288D + 2 mM F6P 0.40 ±0.04 1.4 ± 0.1 59.5 ± 3.2
+ 2 mM Pyruvate 0.45 ± 0.02 2.3 ± 0.3 54.9 ± 1.7
Verification Gel Kinetics Data for P288D Ag.t ADPG PPase
Figure 3. SDS-PAGE of samples P288D mutant purification: 10%
Resolving Gel, 5% Stacking Gel, Electrode Buffer (0.025 M Tris,
0.192 M Glycine, 1% SDS, pH 8.3), 1.5 hours, 100 V, M –
Molecular Weight Marker, 1 – crude extract (~15 µg), 2 – Heat
step/(NH4)2SO4 (~13 µg), 3 – Phenyl Sepharose (~6 µg), 4 – Uno
Q (~2µg), 5 – Blue A (~2 µg).
Table 2. Kinetic Data for WT, P288D and P288E Ag. t. ADPG PPase including S0.5 (mM) and
Hill numbers for the substrates ATP, Vmax (U/mg) values are shown from ATP saturation. The
data was determined using ADPG synthesis direction assays; the assays were performed in
presence of 100 mM HEPES pH 8.0, 0.5 mg/ml BSA, and saturating concentrations of the non-
varied substrate or co-factor (i.e. 2 mM ATP, 10 mM MgCl2, and 0.5 [14C] glucose-1-
phosphate).
M 1 2 3 4 5 6
22
97 kDa
66
45
31
50 kDa
•Previous molecular and kinetic data for P288D showed desensitization to both
inhibitor and activators; however, in the presence of no activators P288D was
determined to have a ~2.5 fold increase in S0.5 values for ATP when compared to
the wild type resulting in a ~4 fold increase in Vmax.
•In order to determine-function relationships, P288D was successful expressed
and purified to near homogeneity in preparation for future crystallization trials and
size exclusion chromatography
•Kinetic analysis of purified E304D enzyme showed:
oActivation by Fructose-1,6-biphosphate, although WT is insensitive (A0.5
=0.40mM, 2.34 fold-activation)
oExhibited A0.5 values for fructose-6-phophate (F6P) and pyruvate similar to
WT
oExhibited ~6 fold and ~3 fold activation lower than WT for F6P and
pyruvate respectively
•Kinetic analysis of purified E304A enzyme showed:
oExhibited 8.1 µM (fold activation 4.18) and 10 µM (4.24 fold activation) A0.5
values for F6P and pyruvate respectively
oExhibited A0.5 values ~10 fold lower than WT indicating higher apparent
affinity
oExhibited fold activation 3 and 2 fold lower then WT for F6P and pyruvate
respectively
•Crystallization trials of the P288D altered protein as a first step toward
solving the three-dimensional structure for comparison to WT
•Size-exclusion chromatography for further purification and determination of
the aggregation state of P288D and other Ag.t. altered proteins for
comparison to WT
•Generation of different mutations derived from the loop region for further
investigation of allosteric regulation mechanism
•Truncation, shortening the loop region, to determine structure-function of
ADPG PPase and importance of the loop region in allosteric regulation
Meyer Lab Team
Department of Chemistry and Biochemistry,CSUF
STEM2 (Supported by the Department of Education)
A0.5(µM)
F6P Hill #
Fold
Activation Pyr. Hill #
Fold
activation FBP Hill #
WT 130 ± 10 2.0± 0.3 12 100 ± 10 1.9 ± 0.3 8.5 N/A N/A N/A
E304A 8.9 ± 1.1 1 4.18 90.0 ± 2.3 1 3.24 N/A N/A N/A
E304D 43.5 ± 19.7 1 3.69 170.2 ± 45.5 1 2.66 402.8 ± 38.9 2 2.34
Kinetics Data for E304A and E304D Ag.t ADPG PPase
Fold
Activation
0 100 200 300 400 500
Activator (æM)
0
4
8
12
Fold
-Act
ivat
ion
WT+F6P
+Pyr
0 50 100 150
Activator (æM)
0
1
2
3
4
Fold
-Act
ivat
ion
E304D+F6P
+Pyr
0 100 200 300 400 500
Activator (æM)
0
2
4
6
Fold
-Act
ivat
ion
E304A
+F6P
+Pyr
Activator
(mM)
Activator
(mM)
Activator
(µM) Activator
(µM) Activator
(µM)
Fo
ld a
ctivatio
n
Fo
ld a
ctivatio
n
Fo
ld a
ctivatio
n
Activator Saturation Plots for E04A and E304D Ag.t ADPG PPase
Table 3. Kinetic Data for WT, E304A, and E304D Ag.t ADPG PPase including A0.5 (µM) and Hill numbers for the activators F6P, Pyr., and FBP and fold activation
values are shown. The data was determined using ADPG synthesis direction assays; the assays were performed in presence of 500 mM HEPES pH 8.0, 10 mg/ml
BSA, and saturating concentrations of the non-varied substrate or co-factor (i.e. 50 mM ATP, 200 mM MgCl2, and 11.8 [14C] glucose-1-phosphate).
Figure 4. Activator saturation plots of WT and E304A and E30D Ag.t ADPG PPase depicting folding
activation vs. activator concentration (µM). The assays were performed in presence of 500 mM HEPES
pH 8.0, 10 mg/ml BSA, and saturating concentrations of the non-varied substrate or co-factor (i.e. 50 mM
ATP, 200 mM MgCl2, and 11.8 [14C] glucose-1-phosphate).
Meyer, Christopher, Jennifer Yirsa, Bruce Gott, and Jack Preiss. 1998. A Kinetic Study of
Site-directed Mutants of Escherichia Coli ADP-glucose Pyrophosphorylase: The Role of
Residue 295 in Allosteric Regulation. Archives of Biochemistry and Biophysics. 352, no.
2: 247-254.
Gomez-Casati, Diego, Robert Igarashi, Christopher Berger, Mark Brandt, Alberto Iglesias,
and Christopher Meyer. 2001. Identification of Functionally Important Amino-terminal
Arginines of Agrobacterium Tumefaciens ADP-glucose Pyrophosphorylase by Alanine
Scanning Mutagenesis. Biochemistry. 40, no. 34: 10169-10178.
Cupp-Vickery, Jill, Robert Igarashi, Marco Perez, Myesha Poland, and Christopher Meyer.
2008. Structural Analysis of ADP-glucose Pyrophosphorylase from the Bacterium
Agrobacterium Tumefaciens. Biochemistry. 47, no. 15: 4439-4451.
Ballicora, Miguel, Alberto Iglesias, and Jack Preiss. 2004. ADP-glucose
Pyrophosphorylase: A Regulatory Enzyme for Plant Starch Synthesis. Photosynthesis
Research. 79, no. 1: 1-24.