| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
|
Plant Physiol. (1998) 117: 989-996 Mutagenesis of the Glucose-1-Phosphate-Binding Site of Potato Tuber ADP-Glucose Pyrophosphorylase1
Department of Biochemistry, Michigan State University, East Lansing, Michigan 48824
Lysine (Lys)-195 in the homotetrameric ADP-glucose pyrophosphorylase (ADPGlc PPase) from Escherichia coli was shown previously to be involved in the binding of the substrate glucose-1-phosphate (Glc-1-P). This residue is highly conserved in the ADPGlc PPase family. Site-directed mutagenesis was used to investigate the function of this conserved Lys residue in the large and small subunits of the heterotetrameric potato (Solanum tuberosum) tuber enzyme. The apparent affinity for Glc-1-P of the wild-type enzyme decreased 135- to 550-fold by changing Lys-198 of the small subunit to arginine, alanine, or glutamic acid, suggesting that both the charge and the size of this residue influence Glc-1-P binding. These mutations had little effect on the kinetic constants for the other substrates (ATP and Mg2+ or ADP-Glc and inorganic phosphate), activator (3-phosphoglycerate), inhibitor (inorganic phosphate), or on the thermal stability. Mutagenesis of the corresponding Lys (Lys-213) in the large subunit had no effect on the apparent affinity for Glc-1-P by substitution with arginine, alanine, or glutamic acid. A double mutant, SK198RLK213R, was also obtained that had a 100-fold reduction of the apparent affinity for Glc-1-P. The data indicate that Lys-198 in the small subunit is directly involved in the binding of Glc-1-P, whereas they appear to exclude a direct role of Lys-213 in the large subunit in the interaction with this substrate.
ADPGlc PPase (EC 2.7.7.27) catalyzes an important regulatory
step in the biosynthesis of starch in plants and of glycogen in
bacteria (Preiss, 1988 Studies based on a wide range of sources have shown that ADPGlc PPase
is regulated by effectors derived from the dominant carbon-assimilation
pathway in the organism. The enzyme from higher plants (Preiss, 1988 The potato (Solanum tuberosum L.) tuber ADPGlc PPase
consists of two different subunits of 51 and 50 kD (Okita et al.,
1990 Chemical modification and site-directed mutagenesis studies have
determined that Lys-195 in ADPGlc PPase from E. coli is
involved in binding of the substrate Glc-1-P (Parsons and Preiss, 1978 Reagents
Bacterial Strains and Media Escherichia coli strain TG1 (F traD36
lacIq  [lacZ]M15
proA+B+/supE
[hsdM-mcrB]5[rk mkMcrB]
thi [lac-proAB]) was used for site-directed
mutagenesis. E. coli mutant strain AC70R1-504 (Carlson et
al., 1976 ), which exhibited negligible ADPGlc PPase activity, was used
for expression of the potato tuber ADPGlc PPase gene (Ballicora et al.,
1995). Both E. coli strains were grown in Luria-Bertani
medium.
Site-Directed Mutagenesis For mutagenesis, the gene for the small subunit of potato tuber ADPGlc PPase was subcloned as an EcoRI fragment from pML 10, a plasmid containing the cDNA gene of the small subunit (Ballicora et al., 1995), into the EcoRI site of M13mp18RF. The gene for the large subunit was subcloned as an XbaI fragment from
pMON17336, a plasmid containing the cDNA gene of the large subunit
(Iglesias et al., 1993; Ballicora et al., 1995), into the
XbaI site of M13mp19RF. For the small subunit after
mutagenesis, the mutated EcoRI fragment was exchanged with
the unmutated EcoRI fragment in pML 10; for the large
subunit, the mutated XbaI fragment was exchanged with the
unmutated XbaI fragment in pMON17336. Site-directed
mutagenesis experiments were performed according to a previously
described method (Sayers et al., 1988) using the in vitro site-directed mutagenesis kit from Amersham. Three heterotetrameric mutant enzymes with a single substitution of Arg, Ala, and Glu at Lys-198 of the small
subunit were designated as
SK198RLwt,
SK198ALwt, and SK198ELwt, respectively.
The mutant enzymes with the substitution of Arg, Ala, and Glu at
Lys-213 of the large subunit were designated as
SwtLK213R,
SwtLK213A, and
SwtLK213E, respectively.
The double-mutant enzyme, in which both Lys-198 of the small subunit
and Lys-213 of the large subunit were replaced with Arg, was designated
as SK198RLK213R. The
oligonucleotides used to create the desired mutations are shown in
Figure 1. Before expression of the mutant enzymes, the entire coding regions of these mutant alleles were sequenced to verify that there were no undesired mutations.
Expression and Purification of Mutant and Wild-Type Enzymes The single mutant enzymes were obtained by co-expressing the mutated plasmid pML 10 (or pMON17336) with unmutated plasmid pMON17336 (or pML 10) in E. coli mutant strain AC70R1-504. The double-mutant enzyme was obtained by co-expressing the two mutated plasmids in AC70R1-504. Mutant enzyme SK198RLwt was expressed as described previously (Ballicora et al., 1995). The other mutant and
wild-type enzymes were expressed in the same manner except that the
concentration of isopropyl- -D-thiogalactopyranoside was
increased from 10 µM to 0.5 mM for induction.
An improved procedure over the one in the previous study (Ballicora et
al., 1995) was used for the purification of the wild-type
and mutant enzymes. In the
hydrophobic-interaction-chromatography step, the enzyme was loaded onto
the column in the presence of 1.2 M ammonium sulfate
instead of 1.3 M potassium phosphate buffer. After the heat
treatment step, a 50% saturation ammonium-sulfate-precipitation step
was added, after which the pellet was dissolved in a minimal volume of
extraction buffer (100 mM Hepes-NaOH, pH 8.0, 5 mM MgCl2, 1 mM EDTA, and
20% [w/v] Suc) and dialyzed against the same buffer overnight. The
dialyzed enzyme was centrifuged at 20,000g for 15 min at
4°C to remove insoluble material before being loaded onto the DEAE
Fractogel column (EM Separations Technology, Gibbstown, NJ).
Assay of ADPGlc PPase Assay I In the pyrophosphorolysis direction, enzyme activity was assayed according to a previously described method (Morell et al., 1987). The
reaction mixture contained 80 mM glycyl-Gly, pH 8.0, 2 mM ADPGlc, 5 mM MgCl2, 3 mM DTT, 2 mM 32PPi
(1000-2000 cpm nmol1), 3 mM 3PGA,
10 mM NaF, 200 µg mL1 BSA, and
enzyme in a total volume of 250 µL. The assay conditions for the
mutant enzymes were identical to those for the wild-type enzymes except
that the amounts of MgCl2 and ADPGlc were altered for some mutant enzymes to obtain maximal activity. The amount of
MgCl2 was increased to 10 mM for
SK198ALwt and
SK198ELwt, and to 20 mM for
SK198RLwt and
SK198RLK213R. For the
SK198ELwt and SK198RLwt enzymes, 3 mM ADPGlc was used.
Assay II In the ADPGlc-synthesis direction, enzyme activity was measured according to a previously described method (Preiss et al., 1966). The
reaction mixture contained 100 mM Hepes-NaOH, pH 8.0, 0.5 mM [U-14C]Glc-1-P (1000-3000 cpm
nmol1), 1.5 mM ATP, 5 mM MgCl2, 3.0 mM 3PGA, 3 mM DTT, 200 µg mL1 BSA, 0.3 unit
of inorganic pyrophosphatase, and enzyme in a final volume of 200 µL.
For assay of the
SK198RLwt,
SK198ALwt,
SK198ELwt, and
SK198RLK213R mutant
enzymes, [8-14C]ATP (about 200-500 cpm
nmol1) instead of
[14C]Glc-1-P was used to monitor the synthesis
of ADPGlc, and the amounts of Glc-1-P and MgCl2
were increased to 30 and 20 mM, respectively, to
obtain maximal activity. For the double-mutant
SK198RLK213R enzyme, the
3PGA concentration was increased to 10 mM in addition to
the changes mentioned above. Because of the high content of Glc-1-P in
the assay mixture, the time for the alkaline phosphatase digestion was
extended to overnight. Control experiments showed that the product,
[14C]ADPGlc, was stable during the overnight
digestion.
Kinetic Studies For determination of kinetic parameters, the concentration of the substrate or effectors tested was systematically varied with the other substrates and effectors fixed at a saturating concentration, as described for assays I and II. Kinetic data were plotted as initial velocity versus substrate or effector concentration. The kinetic constants A0.5, S0.5, and I0.5, which correspond to the concentration of activator, substrate, or inhibitor giving 50% of maximal activation, velocity, or inhibition, respectively, as well as the interaction coefficient nH were obtained from a computer program using a nonlinear, iterative, least-squares fitting to a modified Michaelis-Menten equation (Canellas and Wedding, 1980).
Sugar-Phosphate Specificity Reactions were performed in the ADPGlc-synthesis direction (assay II) with different sugar phosphates substituted for Glc-1-P. [8-14C]ATP (about 200-500 cpm nmol1) was used to monitor the assay. The
reaction time was extended to 30 min at 37°C. Under the conditions
with Glc-1-P as the substrate, the production of ADPGlc was shown to be
linear for both wild-type and mutant enzyme
SK198ALwt. The time for the
alkaline phosphatase digestion was extended to 48 h. The extent of
the reaction was controlled by varying the amount of enzyme used for
the assay.
Thermal Stability Enzyme samples were diluted to give the same final protein concentration (0.3 mg mL1) in a final volume of
20 µL. Dilution buffer was 50 mM Hepes-NaOH, pH 8.0, 5 mM MgCl2, 1 mM EDTA, 20%
(w/v) Suc, and 1 mg mL1 BSA. The enzyme samples
were heated for 5 min in a water bath equilibrated at 60°C, then
immediately placed on ice. The enzyme activities were assayed in the
ADPGlc-synthesis direction, as described in "Assay II."
Protein Assay Protein concentration was measured using the Pierce bicinchoninic acid reagent (Smith et al., 1985) with BSA as the standard.
Protein Electrophoresis and Immunoblotting SDS-PAGE was performed according to the method of Laemmli (1970)
on 10% polyacrylamide slab gels. After electrophoresis, proteins on
the gel were visualized by staining with Coomassie brilliant blue R-250
or electroblotted onto a nitrocellulose membrane (Burnette, 1981).
After electroblotting the nitrocellulose membrane was treated with
affinity-purified rabbit anti-spinach leaf ADPGlc PPase IgG, and the
antigen-antibody complex was visualized by treatment with alkaline
phosphatase-linked goat anti-rabbit IgG followed by staining with
purple alkaline phosphatase-substrate precipitating reagent (Boehringer
Mannheim).
Expression and Purification of Mutant Enzymes Wild-type and mutant enzymes of potato tuber ADPGlc PPase were identified by immunoblotting with antibody prepared against the spinach leaf ADPGlc PPase. It has been shown that the small subunit of the potato tuber enzyme cross-reacts significantly with the spinach leaf ADPGlc PPase antibody (Okita et al., 1990). Under the same
conditions the expression level of all of the mutant enzymes was
similar to that of the wild type based on the results of
immunoblotting. The apparent sizes of these mutant polypeptides were
the same as that of the wild type, having a molecular mass of about 50 kD. Mutant enzyme SK198RLwt
was purified to more than 85% homogeneity, as estimated from about 4 µg of protein electrophoresed on SDS-PAGE. All of the other enzymes
were purified to greater than 95% homogeneity.
Kinetic Characterization of SK198Lwt Mutant Enzymes The apparent affinity for Glc-1-P decreased dramatically when Lys-198 in the small subunit was mutated to either Arg, Ala, or Glu. The S0.5 values for Glc-1-P of the SK198RLwt, SK198ALwt, and SK198ELwt enzymes were about 135-, 400-, and 550-fold higher, respectively, than that of the wild-type enzyme (Table I). Substitution of Lys-198 in the small subunit by Ala and Glu resulted in such large S0.5 changes that they could not be accurately determined (Fig. 2A). The highest Glc-1-P amount used in the assay mixture was 40 mM, which is about two times higher than the S0.5 of the Ala mutant and slightly higher than the S0.5 of the Glu mutant, due to a solubility problem. The values for nH were changed from 1.1 for the wild type to 1.3 to 1.8 for the mutant enzymes. The changes of the Vmax value in the synthesis direction were 2-fold or less, and in the pyrophosphorolysis direction, they were less than 4-fold, except for mutant SK198ELwt. This suggests that Lys-198 in the small subunit has virtually no role in the rate-determining step of catalysis. This is consistent with the observation on the corresponding Lys-195 of the E. coli ADPGlc PPase (Hill et al., 1991). Replacement with Glu not only caused
the largest increase of S0.5 value for
Glc-1-P, but also substantially decreased the catalytic efficiency
relative to the wild-type enzyme.
Kinetic Characterization of SwtLK213 Mutant
Enzymes
Kinetic Characterization of SK198RLK213R Mutant Enzyme When both Lys-198 in the small subunit and Lys-213 in the large subunit were replaced with Arg, the S0.5 value for Glc-1-P was about 100-fold higher than that of the wild-type enzyme (Table I). Considering the 135-fold increase of the S0.5 value in mutant enzyme SK198RLwt, the double mutation did not cause a further decrease in the apparent affinity of Glc-1-P over the single mutation. In either direction of the assay, the Vmax of the double-mutant enzyme was essentially the same as that of the single-mutant enzyme, SK198RLwt.Sugar-Phosphate Specificity Because Lys-198 on the small subunit was implicated in Glc-1-P binding, it was of interest to determine whether the specificities for the substrates of the SK198ALwt mutant enzymes had been changed. To analyze systematically the contribution of each specific hydroxyl group in the binding of substrate to the active site of wild-type and mutant enzymes, a variety of compounds were used in which sugar moieties differ from Glc stereochemically or by substitution or elimination of hydroxyl groups at different positions. In the measurement of the activity of mutant enzyme SK198ALwt, large amounts of enzyme had to be used to obtain accurate measurements. The results are summarized in Table III. When 6-deoxy-Glc-1-P and 6F-Glc-1-P were used to substitute for Glc-1-P, the activity of the wild-type and SK198ALwt enzyme decreased 3.1- to 4.5-fold and 17- to 34-fold, respectively. When the other analogs were used to substitute for Glc-1-P, the activity of the wild-type and SK198ALwt enzymes decreased more than 14- and 150-fold, respectively. Thus, both enzymes showed the largest tolerance toward the elimination or substitution of the hydroxyl group at C-6 of the Glc molecule, followed by the changes at C-2, C-3, and C-4. In no case was there a substrate analog that showed a significantly enhanced reactivity with the mutant enzyme compared with the wild type. Substitution of a carboxyl group at C-6 indicates that GlcUA-1-P is not a substrate. The sugar-phosphate analogs also showed the same pattern of effect for the mutant enzyme SK198RLwt (data not shown). The results indicate that the hydroxyl groups at C-2, C-3, and C-4 probably played much more important roles than the C-6 hydroxyl group in the binding process. Overall, replacement of Lys-198 with either Ala or Arg did not cause any broadened changes in substrate specificity for sugar-1-phosphate.
Thermal Stability of Wild-Type and Mutant Proteins SK198ALwt, SK198ELwt, SwtLK213A, and SK198RLK213R After heat treatment at 60°C for 5 min, the activity of the wild-type enzyme remained unchanged, whereas the SK198ALwt, SK198ELwt, SwtLK213A, and SK198RLK213R enzymes retained 104%, 67%, 94%, and 105% activity, respectively. Therefore, substitution of Lys-198 with a negatively charged Glu made the protein more susceptible to heat inactivation. Nevertheless, neither residue 198 in the small subunit nor residue 213 in the large subunit was critical for the stability of the native folded state of the potato tuber enzyme.
According to the results presented here, we can conclude that
Lys-198 of the small subunit of potato tuber ADPGlc PPase is primarily
involved in Glc-1-P binding. The 135- to 550-fold increases in the
S0.5 value for Glc-1-P when this residue
was replaced by other amino acids explains the high conservation of
this Lys in plant and bacterial ADPGlc PPases. The Lys residue probably
is required for the proper substrate binding to ADPGlc PPase under physiological concentrations of Glc-1-P. Although Lys-198 is critical in interacting with Glc-1-P, it is obviously not essential for thermal
stability. From the modest effect on Vmax
values and the kinetic constants for ATP, Mg2+,
3PGA, and Pi (Tables I and II), Lys-198 is probably neither involved in
the rate-limiting step of the catalytic mechanism nor responsible for
maintaining the native conformation of the enzyme. The
nH of Glc-1-P of the mutant enzymes was
increased to 1.3 to 1.8, compared with 1.1 for the wild type. However,
because both the SK198ALwt
and SK198ELwt mutant
enzymes had high S0.5 values for Glc-1-P,
it was impossible to perform kinetic studies for them under saturated
concentrations of Glc-1-P. Only 50 to 70%
Vmax could be attained based on the
estimation from the Lineweaver-Burk plot. Therefore, the
nH values could be overestimated for these two enzymes. Nevertheless, it has been observed for some enzymes that a
single mutation causing decreased affinity for a ligand results in an
increase in cooperativity (Stebbins and Kantrowitz, 1992
Received February 18, 1998;
accepted April 20, 1998.
Abbreviations:
ADPGlc PPase, ADP-Glc pyrophosphorylase.
6F-Glc-1-P, 6-fluoro-Glc-1-P.
3-PGA, 3-phosphoglycerate.
We thank Dr. Steven Withers (Department of Chemistry, University
of British Columbia, Vancouver, Canada) for kindly providing the
following sugar phosphates: 6-deoxy-Glc-1-P, 6F-Glc-1-P, 2F-Glc-1-P, 3-deoxy-Glc-1-P, and 3F-Glc-1-P.
Ball S,
Marianne T,
Dirick L,
Fresnoy M,
Delrue B,
Decq A
(1991)
A Chlamydomonas reinhardtii low-starch mutant is defective for 3-phosphoglycerate activation and orthophosphate inhibition of ADP-glucose pyrophosphorylase.
Planta
185:
17-26
Ballicora MA, Fu Y, Nesbitt NM, Preiss J (1998) ADP-glucose
pyrophosphorylase from potato tuber. Site-directed mutagenesis studies
of the regulatory sites. Plant Physiol (in press)
Ballicora MA,
Laughlin MJ,
Fu Y,
Okita TW,
Barry GF,
Preiss J
(1995)
Adenosine 5
Burnette WN
(1981)
"Western blotting": electrophoretic transfer of proteins from sodium dodecyl sulfate-polyacrylamide gels to unmodified nitrocellulose and radiographic detection with antibody and radioiodinated protein A.
Anal Biochem
112:
195-203
[CrossRef][ISI][Medline]
Canellas PF,
Wedding RT
(1980)
Substrate and metal ion interactions in the NAD+ malic enzyme from cauliflower.
Arch Biochem Biophys
199:
259-264
[Medline]
Carlson CA,
Parsons TF,
Preiss J
(1976)
Biosynthesis of bacterial glycogen: activator-induced oligomerization of a mutant Escherichia coli ADP-glucose synthase.
J Biol Chem
251:
7886-7892
First EA,
Fersht AR
(1993)
Mutation of lysine 233 to alanine introduces positive cooperativity into tyrosyl-tRNA synthetase.
Biochemistry
32:
13651-13657
[CrossRef][Medline]
Hill MA,
Kaufmann K,
Otero J,
Preiss J
(1991)
Biosynthesis of bacterial glycogen: mutagenesis of a catalytic site residue of ADP-glucose pyrophosphorylase from Escherichia coli.
J Biol Chem
266:
12455-12460
Hossain SA,
Tanizawa K,
Kazuta Y,
Fukui T
(1994)
Overproduction and characterization of recombinant UDP-glucose pyrophosphorylase from Escherichia coli K-12.
J Biochem
115:
965-972
Iglesias AA,
Barry GF,
Meyer C,
Bloksberg L,
Nakata PA,
Greene T,
Laughlin MJ,
Okita TW,
Kishore GM,
Preiss J
(1993)
Expression of the potato tuber ADP-glucose pyrophosphorylase in Escherichia coli.
J Biol Chem
268:
1081-1086
Iglesias AA,
Kakefuda G,
Preiss J
(1991)
Regulatory and structural properties of the cyanobacterial ADP-glucose pyrophosphorylase.
Plant Physiol
97:
1187-1195
Jiang XM,
Neal B,
Santiago F,
Lee SJ,
Romana LK,
Reeves PR
(1991)
Structure and sequence of the rfb (O antigen) gene cluster of Salmonella serovar typhimurium (strain LT2).
Mol Microbiol
5:
695-713
[CrossRef][ISI][Medline]
Katsube T,
Kazuta Y,
Mori H,
Nakano K,
Tanizawa K,
Fukui T
(1990)
UDP-glucose pyrophosphorylase from potato tuber: cDNA cloning and sequencing.
J Biochem
108:
321-326
Köplin R,
Arnold W,
Hotte B,
Simon R,
Wang G,
Pühler A
(1992)
Genetics of xanthan production in Xanthomonas campestris: the xanA and xanB genes are involved in UDP-glucose and GDP-mannose biosynthesis.
J Bacteriol
174:
191-199
Laemmli UK
(1970)
Cleavage of structural proteins during the assembly of the head of bacteriophage T4.
Nature
227:
680-685
[CrossRef][Medline]
Marolda CL,
Valvano MA
(1993)
Identification, expression, and DNA sequence of the GDP-mannose biosynthesis genes encoded by the O7 rfb gene cluster of strain VW187 (Escherichia coli O7:K1).
J Bacteriol
175:
148-158
May TB,
Shinabarger D,
Boyd A,
Chakrabarty AM
(1994)
Identification of amino acid residues involved in the activity of phosphomannose isomerase-guanosine 5
Morell MK,
Bloom M,
Knowles V,
Preiss J
(1987)
Subunit structure of spinach leaf ADP-glucose pyrophosphorylase.
Plant Physiol
85:
182-187
Okita TW,
Nakata PA,
Anderson JM,
Sowokinos J,
Morell M,
Preiss J
(1990)
The subunit structure of potato tuber ADP-glucose pyrophosphorylase.
Plant Physiol
93:
785-790
Parsons TF,
Preiss J
(1978)
Biosynthesis of bacterial glycogen: incorporation of pyridoxal phosphate into the allosteric activator site and an ADP-glucose protected pyridoxal phosphate binding site of Escherichia coli B ADP-glucose synthase.
J Biol Chem
253:
6197-6202
Preiss J
(1988)
Biosynthesis of starch and its regulation.
In
J Preiss,
eds, The Biochemistry of Plants, Vol 14.
Academic Press, San Diego, CA, pp 181-254
Preiss J
(1991)
Biology and molecular biology of starch synthesis and its regulation.
In
BJ Miflin,
eds, Surveys of Plant Molecular and Cell Biology, Vol 7.
Oxford University Press, Oxford, UK, pp 59-114
Preiss J (1997) Modulation of starch synthesis. In C
Foyer, P Quick, eds, Engineering Improved Carbon and Nitrogen Resource
Use Efficiency in Higher Plants. Taylor and Francis, London, pp 81-104
Preiss J,
Romeo T
(1989)
Physiology, biochemistry and genetics of bacterial glycogen synthesis.
Adv Microb Physiol
30:
183-238
[Medline]
Preiss J,
Shen L,
Greenberg E,
Gentner N
(1966)
Biosynthesis of bacterial glycogen: activation and inhibition of the adenosine diphosphate glucose pyrophosphorylase of Escherichia coli B.
Biochemistry
5:
1833-1845
[CrossRef][Medline]
Preiss J, Sivak M (1996) Starch synthesis in sinks and sources.
In E Zamski, A Schaffer, eds, Photoassimilate Distribution
in Plants and Crops. Marcel Dekker, New York, pp 63-96
Ragheb JA,
Dottin RP
(1987)
Structure and sequence of a UDP glucose pyrophosphorylase gene of Dictyostelium discoideum.
Nucleic Acids Res
15:
3891-3906
Sanwal GG,
Preiss J
(1967)
Biosynthesis of starch in Chlorella pyrenoidosa. II. Regulation of ATP:alpha-D-glucose 1-phosphate adenyl transferase (ADP-glucose pyrophosphorylase) by inorganic phosphate and 3-phosphoglycerate.
Arch Biochem Biophys
119:
454-459
[CrossRef][Medline]
Sayers JR,
Schmidt W,
Eckstein F
(1988)
5
Smith PK,
Krohn RI,
Hermanson GT,
Mallia AK,
Garter FH,
Provenzano MD,
Fujimoto EK,
Goeke NM,
Olson BJ,
Kenk DK
(1985)
Measurement of protein using bicinchoninic acid.
Anal Biochem
150:
76-85
[CrossRef][ISI][Medline]
Smith-White B,
Preiss J
(1992)
Comparison of proteins of ADP-glucose pyrophosphorylase from diverse sources.
J Mol Evol
34:
449-464
[CrossRef][ISI][Medline]
Stebbins JW,
Kantrowitz ER
(1992)
Conversion of the noncooperative Bacillus subtilis aspartate transcarbamoylase into a cooperative enzyme by a single amino acid substitution.
Biochemistry
31:
2328-2332
[CrossRef][Medline]
Thorson JS,
Kelly TM,
Liu HW
(1994)
Cloning, sequencing, and overexpression in Escherichia coli of the
Varnó D,
Boylan SA,
Okamoto K,
Price CW
(1993)
Bacillus subtilis gtaB encodes UDP-glucose pyrophosphorylase and is controlled by stationary-phase transcription factor sigma B.
J Bacteriol
175:
3964-3971
Weber H,
Heim U,
Borisjuk L,
Wobus U
(1995)
Cell-type specific, coordinate expression of two ADPGlc pyrophosphorylase genes in relation to starch biosynthesis during seed development of Vicia faba L.
Planta
195:
352-361
[ISI][Medline]
This article has been cited by other articles:
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Top
Abstract
Introduction
-1,4-glucan synthesis. ADPGlc PPase from higher plants is
heterotetrameric and encoded by two different genes (Smith-White and
Preiss, 1992
),
SK198RLwt(
),
SK198ALwt(
), and
SK198ELwt(
), 100% activity corresponds to
1.2, 16.6, 33.7, and 39.0 nmol 10 min
), 100%
activity corresponds to 1.2 and 1.6 nmol 10 min
-amino group
and the negatively charged phosphate group of Glc-1-P. Those hydroxyl
groups may interact with the side chains of the other residues in the
active site, i.e. by hydrogen bonding, to anchor the substrate
correctly. Therefore, those analogs tested would have similar effects
on the wild-type as well as the mutant enzymes.
