From the Department of Biochemistry, Biophysics, and Molecular Biology, Iowa State University, Ames, Iowa 5011
Received for publication, October 22, 2002
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ABSTRACT |
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Vertebrates have acidic and basic isozymes of
adenylosuccinate synthetase, which participate in the first committed
step of de novo AMP biosynthesis and/or the purine
nucleotide cycle. These isozymes differ in their kinetic properties and
N-leader sequences, and their regulation may vary with tissue type.
Recombinant acidic and basic synthetases from mouse, in the presence of
active site ligands, behave in analytical ultracentrifugation as
dimers. Active site ligands enhance thermal stability of both isozymes.
Truncated forms of both isozymes retain the kinetic parameters and the
oligomerization status of the full-length proteins. AMP potently
inhibits the acidic isozyme competitively with respect to IMP. In
contrast, AMP weakly inhibits the basic isozyme noncompetitively with
respect to all substrates. IMP inhibition of the acidic isozyme is
competitive, and that of the basic isozyme noncompetitive, with respect
to GTP. Fructose 1,6-bisphosphate potently inhibits both isozymes competitively with respect to IMP but becomes noncompetitive at saturating substrate concentrations. The above, coupled with structural information, suggests antagonistic interactions between the active sites of the basic isozyme, whereas active sites of the acidic isozyme
seem functionally independent. Fructose 1,6-bisphosphate and IMP
together may be dynamic regulators of the basic isozyme in muscle,
causing potent inhibition of the synthetase under conditions of high
AMP deaminase activity.
Adenylosuccinate synthetase (IMP:L-aspartate ligase
(GDP-forming), EC 6.3.4.4) is present in almost all organisms, the only exceptions being some intracellular prokaryotic parasites (1). Adenylosuccinate synthetases are well conserved through evolution, exhibiting, for instance, ~40% sequence identity between eubacteria and mammals (2, 3). Eukaryotic synthetases differ from their prokaryotic counterparts by the presence of N-terminal leader sequences
(~30 residues) and by truncations (~10 residues) at their C termini
(2-4). The synthetase catalyzes the first committed step in the
de novo biosynthesis of AMP from IMP and, in vertebrates, is
also a component of the purine nucleotide cycle
(PNC)1 (2, 5-7). Mammals
have two different synthetase isozymes (2, 3, 7-11). The basic isozyme
(hereafter AdSS1) has a higher Km value for IMP and
a lower Km value for L-aspartate than
the acidic form (AdSS2). AdSS2 is less susceptible to inhibition by
fructose 1,6-bisphosphate (Fru-1,6-P2) than AdSS1 but more
strongly inhibited by nucleotides (7, 12). On the basis of these
findings alone, investigators assigned AdSS2 to de novo
biosynthesis of AMP and AdSS1 to the PNC (2, 6, 12).
The PNC is active in muscle, brain, kidney, liver, and pancreatic
islets (5, 13-17) and involves adenylosuccinate synthetase, adenylosuccinate lyase, and AMP deaminase in the following net reaction
(Reaction 1):
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
The PNC may have multiple roles as follows: 1) shifting the
adenylate kinase equilibrium in the direction of ATP formation by
converting AMP into IMP; 2) liberating ammonia from amino acids by
using L-aspartate as a donor; 3) regulating glycolysis (IMP activates glycogen phosphorylase and both AMP and ammonia activate phosphofructokinase); and 4) providing Krebs cycle intermediates (fumarate) in tissues that lack pyruvate carboxylase (2, 6).
The impact of the PNC on the metabolism of various tissues is unsettled (18-21), as is the assignment of the two isozymes to mutually exclusive metabolic roles (18). Moreover, in muscle, where exercise can increase IMP concentration up to 50-fold, the PNC may work asynchronously; AMP deaminase works only when AdSS1 and/or adenylosuccinate lyase is quiescent (3, 22-25). During recovery (restoration of basal IMP levels) AMP deaminase is inactive (24, 26). Indeed, interactions with myosin activate AMP deaminase, a process regulated by the decrease in the ATP concentration during exercise (24, 26). No study has demonstrated regulation of AdSS1 in the context of the PNC, although slight inhibition of the basic isozyme occurs at high concentrations of IMP (8, 13, 27).
Mouse recombinant AdSS1 has a significantly lower Km for IMP than that reported for the basic isozyme isolated from either rat or rabbit (28). Reported Km values for adenylosuccinate synthetases vary considerably (2, 6, 28) due, in part, to variations in assay protocols and conditions of assay, as well as intrinsic differences in the synthetases themselves (2, 6). The low natural abundance of AdSS2 has been an impediment to its purification and rigorous evaluation (7, 9, 18). Preparations of AdSS2 from malignant cells, such as Novikoff ascites tumor cells (29) and Yoshida sarcoma tumor cells (30), have provided, save in one instance (30), specific activities significantly lower than that of AdSS1 (2, 6). Human and mouse AdSS2 have been cloned (3, 4) and the latter overexpressed in COS (African green monkey kidney) cells, but no kinetic characterization was reported (3). Native states of oligomerization for each isozyme remain ambiguous, as reports of monomeric and dimeric AdSS1 and AdSS2 are in the literature (8, 10, 29-31). In contrast, the synthetase from Escherichia coli is active as a dimer (32, 33) and exists in a monomer-dimer equilibrium (34).
Reported here are first instances of heterologous overproduction and
kinetic characterization of mouse AdSS2. The
kcat values for recombinant AdSS2 and AdSS1 are
almost identical. AdSS2, relative to AdSS1, has a slightly lower
Km for IMP and GTP and a significantly higher
Km for L-aspartate. High (but physiologically relevant) concentrations of IMP inhibit AdSS1 but not
AdSS2. Adenylosuccinate, GDP, and GMP are strong inhibitors of both
isozymes, but AMP, which potently inhibits AdSS2, is a weak inhibitor
of AdSS1. Furthermore, the kinetic mechanism of AMP inhibition differs
for the two isozymes. Fru-1,6-P2 might be a physiologically
significant inhibitor of AdSS1 but not of AdSS2. Truncated isozymes
(N-terminal leader sequence removed) retain the kinetic properties and
state of oligomerization of their full-length counterparts. Mouse
isozymes exhibit a monomer-dimer equilibrium, and both GTP and IMP
stabilize the dimer. Differences in mouse synthetases reported here do
not support mutually exclusive metabolic roles for the two isozymes.
Moreover, our findings support the asynchronous operation of the PNC in muscle.
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EXPERIMENTAL PROCEDURES |
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Materials-- E. coli strain BL21 (DE3), plasmid pET28b, nickel-nitrilotriacetic acid-agarose, and the thrombin cleavage capture kit were from Novagen, Inc. Restriction enzymes, DNA ligase, and Vent Polymerase were from New England Biolabs. All other reagents were from Sigma unless noted otherwise.
Construction of Full-length and Truncated Synthetases-- The cloning of full-length AdSS1 into the expression plasmid pET28b was described previously (28). cDNA for mouse acidic adenylosuccinate synthetase (AdSS2) was kindly provided by Dr. F. B. Rudolph (Department of Biochemistry and Cell Biology, Rice University, Houston, TX) as a pSPORT1clone (11). A fragment of 1371 bp was amplified using the following primers: forward, 5'-CCCTTGTCATATGTCGATCTCCGAGAGCAGC-3' (NdeI restriction site underlined), and reverse, 5'-CCGCTCGAGTTAGAAGAGCTGAATCATGGACTC-3' (XhoI restriction site underlined). Insertion of the amplified fragment into corresponding sites of the pET28b expression vector resulted in the plasmid pAdSS2a. An NdeI restriction site located into the AdSS2 open reading frame was removed by a silent mutation. Truncated AdSS1 (AdSS1-Tr) and AdSS2 (AdSS2-Tr) were generated using the forward primers 5'-CCCTTGTCATATGACTGGCTCTCGCGTGACCGTG-3' and 5'-CCCTTGTCATATGGGGAACCGGGTGACTGTGGTG-3', respectively (NdeI restriction sites underlined). All constructs were checked by sequencing (Iowa State University DNA sequencing facility).
Sequence Alignments-- DNA and protein sequences were aligned using Multalign (35). Published sequences of mouse AdSS2 (GenBankTM gi, 404056 and 6671520) (11) were compared with other full-length sequences from mouse (GenBankTM gi: 12845574, 20831732, and 12836391) and expressed sequence tags (GenBankTM gi: 3519613, 12089636, 3521474, and 1738606). The published sequence of human AdSS2 (GenBankTM gi: 415848) (4) was compared with other (redundant) sequences (GenBankTM gi: 10438053 and 15214462). The sequence for human AdSS1 came from available full-length clones and expressed sequence tags (GenBankTM gi: 18583312, 16549233, 12889641, 12901898, 16181164, 13408903, 14817011, and 14676160).
Expression and Purification of Recombinant Isozymes-- AdSS1 was produced and purified as described previously (28). The same procedure was used for AdSS1-Tr. AdSS2 was expressed in E. coli BL21 (DE3) at 37 °C in LB media containing 30 µg/ml kanamycin. Cells were collected by centrifugation (4,000 × g for 10 min at 4 °C), resuspended in lysis buffer (50 mM NaH2PO4, 300 mM NaCl, 10 mM imidazole, pH 8, and 1 mM phenylmethanesulfonyl fluoride), and then disrupted by sonication. After centrifugation (24,000 × g for 30 min), the supernatant was loaded onto a nickel-nitrilotriacetic acid-agarose column, previously equilibrated with 50 mM NaH2PO4, 300 mM NaCl, 10 mM imidazole, pH 8. The column was washed sequentially with 10 volumes of each of the three buffers, differing from the above only in the concentration of imidazole (20, 30, and then 40 mM). Bound protein eluted with 300 mM imidazole. After dialysis in 50 mM HEPES, 50 mM NaCl, 1 mM dithiothreitol, and 0.5 mM EDTA, pH 7.0, the enzyme was loaded at 0.5 ml/min onto a DEAE-Sepharose column, equilibrated with dialysis buffer. AdSS2 was eluted by a linear gradient (0-200 mM NaCl). AdSS2-Tr was overproduced and purified as above with one modification; cell cultures were maintained at 25 °C after induction. At 37 °C the truncated protein appears in inclusion bodies. Removal of the polyhistidyl tag employed the thrombin cleavage capture kit. Recombinant E. coli adenylosuccinate synthetase was overproduced and purified as described elsewhere (34).
Enzyme Assay--
Protein concentration was determined by the
method of Bradford (36), using bovine serum albumin as a standard.
Enzyme activity was determined at an absorbance of 280 nm and at
22 °C as described previously (37). A standard assay buffer for
AdSS1 contained 40 mM HEPES, pH 6.7, 8 mM
magnesium acetate, 150 µM GTP, 250 µM IMP,
and 2 mM aspartate. For AdSS2, the assay buffer contained 40 mM HEPES, pH 6.7, 8 mM magnesium acetate,
150 µM GTP, 200 µM IMP, and 8 mM
L-aspartate. The reaction was started by the addition of up to 1 µg/ml enzyme. Under these conditions the reaction was linear for 1 min. The Hill coefficient for Mg2+ was determined by
varying the concentration of magnesium acetate from 0.2 to 4 mM and from 0.05 to 2 mM for AdSS1 and AdSS2,
respectively. Ki values for Fru-1,6-P2,
AMP, GDP, and GMP were determined by holding two substrates at
saturating levels and varying the concentration of the third substrate
over 18 Km, at different fixed concentrations of
inhibitors ranging over 0.5
2 Ki. In experiments to
determine the Ki of IMP inhibition, concentrations
of GTP varied from 15 to 200 µM, those of IMP ranged from
40 to 4,000 µM, and concentrations of
L-aspartate and Mg2+ were 2 and 8 mM, respectively. Kinetic data were analyzed with the
computer program GraFit (38).
Analytical Ultracentrifugation--
Sedimentation equilibrium
experiments were performed in a Beckman Optima XL-A analytical
ultracentrifuge using an An-60 Ti rotor, rotor speeds of 9,000 and
15,000 rpm, a temperature of 4 °C, and protein concentrations of
0.10.5 mg/ml in 20 mM HEPES, pH 7.2, 20 mM
NaCl, and 1 mM dithiothreitol. Centrifugation in the
presence of ligands (25 µM IMP, 25 µM GTP,
1 µM hadacidin, 2 mM magnesium acetate)
employed only one concentration of protein (0.3 mg/ml). Samples were
centrifuged for 12 h. Equilibration was verified then by 3 scans
recorded at 4-h intervals. Stepwise radial scans were performed at 280 nm, using a step-size of 0.001 cm, with each datum being the average of
30 measurements. Data were analyzed using the "Ideal" model on the
Optima XL-A Analysis software (version 2.0). Partial specific volumes
of 0.741, 0.743, 0.740, and 0.745 ml/g for AdSS1, AdSS1-Tr, AdSS2, and
AdSS2-Tr, respectively, were determined from the amino acid composition and published tables (39). Samples were centrifuged at 40,000 rpm for
15 h to sediment all protein, and then radial scans were recorded
to obtain a base-line correction for each cell. Inclusion of the second
virial coefficient did not improve fits, indicating that nonideality is
not present in the system.
Partial Purification of Mouse Isozymes from Tissue--
Mouse
liver and skeletal muscle were disrupted using a Polytron homogenizer
(Brinkmann Instruments) in a buffer containing 50 mM HEPES,
pH 7.0, 50 mM NaCl, 1 mM dithiothreitol, 0.5 mM EDTA, 1 mM phenylmethanesulfonyl fluoride,
and 15 µg/ml leupeptin. The homogenate was centrifuged at 25,000 × g for 30 min. The recovered supernatant fluid was heated
(60 °C, 1 min) and then centrifuged (25,000 × g, 45 min). The supernatant was subjected to ammonium sulfate fractionation.
Proteins that precipitated in the range of 4060% (w/v) ammonium
sulfate were retained. At this point adenylosuccinate synthetase
activity was readily detected. Precipitated protein was dialyzed
against 25 mM HEPES, 25 mM NaCl, 1 mM dithiothreitol, and 0.5 mM EDTA, pH 7.5, and
then loaded onto a DEAE-Sepharose column equilibrated with the same
buffer. The column retained the acidic but not the basic isozyme. 1 M NaCl washed the acidic isozyme from the column.
Identities of the acidic and basic isozymes were confirmed by Western
blots and their kinetic properties.
Antibodies and Western Blots--
Polyclonal antibodies against
recombinant AdSS1 were raised in rabbits at the Iowa State University
protein facility and were then purified by affinity chromatography,
using an Econo-Pac serum IgG purification kit (Bio-Rad). A Sepharose 4B
column containing immobilized AdSS2 eliminated cross-reacting
antibodies. Protein transfer to a nitrocellulose membrane was performed
as recommended by the manufacturer (Bio-Rad) in a buffer containing 25 mM Tris, pH 8.3, 192 mM glycine, 20% methanol,
and 0.05% SDS. Detection of antigen-antibody complexation employed the
Opti-4CN kit (Bio-Rad).
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RESULTS |
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Sequence Comparison and Validation--
Nucleotide sequences of
mouse AdSS2, reported here and by Guicherit et al. (11),
differ at positions 499 (C in Ref. 11 is now G) and 595 (A becomes G).
As a consequence, Arg167 and Thr199 become
glycine and alanine, respectively, identical to corresponding residues
in human AdSS2 (4). Moreover, Gly167 is invariant among
synthetases from 43 organisms, representing 30 major phylogenetic
lineages. The published sequence of human AdSS2 (4) also differs with
respect to other sequence information; Ala24 should be
arginine and an additional residue (proline) comes after position 24. The N-terminal leader sequence in mouse and human AdSS2 then each have
26 amino acid residues (Fig. 1). Sequence identity between AdSS1 and AdSS2 from the same source (mouse or human)
is ~75% but exceeds 95% between like isozymes from different sources. N-terminal leader sequences of AdSS1 and AdSS2 from the same
source are only 25% identical. In contrast, N-terminal leader sequences between mouse and human AdSS1 are 97% identical, and those
between mouse and human AdSS2 are 73% identical.
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Purity and Yield-- AdSS1-Tr and AdSS2-Tr are shorter by 28 and 26 amino acid residues, respectively, than their full-length counterparts (Fig. 1). The yield of purified AdSS1-Tr was comparable with that of full-length AdSS1 (5-10 mg/liter of cell culture), but substantial quantities of AdSS1 and AdSS1-Tr did appear in inclusion bodies. In contrast, yields of recombinant AdSS2 and AdSS2-Tr were ~ 25 mg/liter of cell culture, ~20% of the total soluble protein. SDS-PAGE of samples revealed a single band of ~50 kDa. Full-length and truncated AdSS2 are stable for several days at 4 °C. AdSS2 is stable with respect to freeze/thaw cycles in buffers supplemented with 30% glycerol. Full-length AdSS1, with or without their N-terminal polyhistidyl tags, have identical kinetic parameters and crystallize under the same conditions (28). Similarly, truncation of the N-terminal polyhistidyl tag from AdSS2 does not change its kinetic properties (data not shown).
Thermal Stability--
In the absence of ligands, the E. coli synthetase is in a monomer-dimer equilibrium
(Kd ~10 µM) (34). The presence of
active site ligands, such as IMP and GTP, significantly increases thermal stability of E. coli synthetase. Moreover, the
thermal stability of the E. coli synthetase increases with
protein concentration.2 In
the absence of ligands, AdSS1 and AdSS2 are more stable than the
E. coli synthetase, but IMP and GTP do not greatly enhance the thermal stability of the mammalian isozymes (Fig.
2). AdSS1-Tr and AdSS2-Tr have the same
thermal stability as their full-length counterparts, ruling out an
effect due to the N-terminal leader sequence.
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Native Molecular Weight of AdSS1 and AdSS2-- Equilibrium sedimentation ultracentrifugation indicates single species of molecular mass 86.0 ± 3.9 and 90.7 ± 2.5 kDa for AdSS1 and AdSS2, respectively. Predicted masses are 101.1 and 100.7 kDa, derived from amino acid sequences of recombinant AdSS1 and AdSS2, respectively. Equilibrium ultracentrifugation of samples in the presence of IMP, GTP, Mg(acetate)2, and hadacidin (N-hydroxy-N-formylglycine, a potent competitive inhibitor with respect to L-aspartate) showed increased molecular masses of 103.6 ± 5.4 and 101.5 ± 4.3 kDa for AdSS1 and AdSS2, respectively. The behavior of AdSS1-Tr and AdSS2-Tr was similar to their full-length counterparts. In the absence of active site ligands, molecular masses were 86.1 ± 5.9 and 84.8 ± 2.7 kDa for AdSS1-Tr and AdSS2-Tr, respectively; and in the presence of substrate/substrate analogues, their molecular masses were 101.1 ± 7.2 and 96.4 ± 0.8 kDa, respectively. Molecular masses of the recombinant mouse isozymes did not change over a protein concentration from 0.1 to 0.5 mg/ml, whereas the molecular mass of the E. coli synthetase varied from 47.8 ± 2.1 to 56.9 ± 2.0 kDa (experiments at 15,000 rpm). Evidently, over the range of protein concentration accessible to analytical centrifugation, AdSS1 and AdSS2 are predominantly dimers, but a small shift from monomer to dimer occurs in the presence of ligands. The Kd value for the mammalian isozymes then, in the absence of ligands, is significantly lower than that of E. coli synthetase (34). The molecular weight (78 kDa) of a yeast synthetase, determined by gel filtration, lies between that of a monomer and dimer (40), whereas plant synthetases are either monomers or dimers, depending on the methodology of mass determination (41).
Km, kcat, pH, and Buffer Effects-- Km values for AdSS1-Tr are comparable with those of AdSS1 (28), but kcat is slightly lower, perhaps due to a small component of misfolded AdSS1-Tr. Relative to AdSS1, AdSS2 has a lower Km for IMP, a similar Km for GTP, and significantly higher Km for L-aspartate (Table I). AdSS2-Tr has a slightly higher Km value for L-aspartate than that of AdSS2. Km values for IMP and L-aspartate of tissue-derived AdSS1 and AdSS2 were similar to those of the recombinant isozymes (data not shown).
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Values of kcat and Km of the
E. coli synthetase are sensitive to pH in the range of
7.07.7 (42). As the pH in exercising muscle can decrease from 6.9 to
6.4, we determined whether changes in pH influence kinetic parameters
of AdSS1 or AdSS2; however, Km values remained
constant from pH 6.5 to 7.2. The pH optima for AdSS1 and AdSS2 lie
between 6.6 and 6.9. As the pH optima are nearly out of the buffering
range of HEPES (pKa 7.5), other more suitable
buffers were tested. MES, MOPS, and PIPES buffers support similar
turnovers for AdSS2 and the E. coli synthetase. In contrast,
AdSS1 in 40 mM MES and PIPES has only 40 and 10%,
respectively, of the specific activity of AdSS1 in HEPES.
Metal Requirement--
AdSS2 and the E. coli synthetase
reach maximum activities in 2 mM Mg(acetate)2,
but AdSS1 requires 8 mM Mg(acetate)2.
Concentrations above 10 mM Mg(acetate)2 are
inhibitory for all systems. Inhibition is more pronounced when
Cl is a counterion to Mg2+ instead of
acetate. Hill coefficients for Mg2+ are 1.1 ± 0.1 for
AdSS1 and 1.0 ± 0.2 for AdSS2. The Hill coefficient for
Mg2+ is 2 for the E. coli synthetase (43).
Crystal structures of AdSS1 (44, 45),
AdSS2,3 and the E. coli synthetase (46, 47), however, reveal only one
Mg2+ per subunit. Evidently, studies of AdSS1 and AdSS2
will not clarify the role of the "second" Mg2+ inferred
by the kinetics of the E. coli synthetase.
Metabolite and Substrate Inhibition-- Mouse synthetases are subject to potent inhibition by adenylosuccinate, GDP, AMP, and GMP (Table II). In contrast to product and GMP inhibition, AMP inhibits AdSS1 and AdSS2 with marked differences. The Ki value for AMP is 12-fold lower for AdSS2 than AdSS1. Moreover, AMP is a competitive inhibitor with respect to IMP for AdSS2 but noncompetitive with respect to IMP for AdSS1.
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Fru-1,6-P2 is the only intermediate of glycolysis that inhibits mammalian adenylosuccinate synthetases at concentrations below 1 mM (1, 2). Fru-1,6-P2 inhibits AdSS1 more potently than AdSS2 in plots of reciprocal velocity against 1/GTP and 1/L-aspartate (6- and 8-fold, respectively), but inhibition of the two isozymes is nearly the same in plots of reciprocal velocity against 1/IMP. Fru-1,6-P2 is a noncompetitive inhibitor with respect to all substrates.
High concentrations of IMP (>1 mM) at a saturating
concentration of GTP (>150 µM) inhibit AdSS1 but not
AdSS2; however, IMP inhibits both isozymes at concentrations of GTP
below its Km value (Fig.
3). Scheme
I represents IMP inhibition of AdSS2 and AdSS1. In the case of AdSS2,
Kii Ki, whereas for AdSS1
Kii must assume a value comparable with that of
Ki in order to observe IMP inhibition in the
presence of saturating concentrations of GTP. In the context of a
saturating concentration of L-aspartate and a rapid
equilibrium Random Bi Bi kinetic mechanism for IMP and GTP (37, 48),
the expression for initial velocity associated with Scheme I is shown
in Equation 1,
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(Eq. 1) |
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(Eq. 2) |
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DISCUSSION |
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E. coli and mouse synthetases exhibit a monomer-dimer equilibrium, in which GTP and IMP stabilize the dimer. Mouse isozymes in the absence of ligands, however, are more stable than the E. coli synthetase on the basis of thermal stability and analytical ultracentrifugation. The monomer-dimer equilibrium may be a property common to all adenylosuccinate synthetases. The dimer is the active form of the E. coli synthetase, and evidently each of its subunits independently achieves maximum velocity in the presence of saturating substrates (32). Indeed, an arginyl side chain (Arg143 in the E. coli synthetase) critical to the recognition of IMP (34) comes to the active site from a symmetry-related subunit of the dimer and is present in all known sequences of synthetase. Hence, regulatory mechanisms that impair subunit dimerization are conceptually possible but have not been demonstrated for any synthetase in vivo. The increased stability of the mouse dimers relative to E. coli dimer could be exploited, however, in the design of drugs (antibiotics) that target the subunit interface.
N-terminal truncations of the mouse isozymes do not alter dimer stability or kinetics. Therefore, the functional differences exhibited by the two isozymes arise from their core sequences, which are ~75% identical. Nevertheless, the N-leader sequences of AdSS1 and AdSS2 diverge significantly, and yet each is conserved across mammalian species. The latter observation suggests possible differences in regulation, protein-protein interactions, and/or intracellular distribution for the two isozymes. The N-leader sequence for the synthetase from plants, for instance, putatively targets it to the chloroplast (49). The basic isozyme binds to F-actin and reconstructed thin microfilaments (50-51), but no hard evidence as yet implicates the N-leader sequence of AdSS1 in these interactions.
Previous studies (8, 13, 27) have documented IMP inhibition of AdSS1, but have disagreed as to whether GTP antagonizes or reinforces the phenomenon (8, 13). Nonetheless, IMP does bind to the GTP pocket of AdSS1 as evidenced by the crystal structure of an IMP complex (44). Furthermore, all residues of the GTP pocket are identical in AdSS1 and AdSS2. The EA2 complex in Scheme I then may well represent IMP molecules bound to the IMP and GTP pockets within the same subunit of a dimer. High levels of GTP would relieve such inhibition, but AdSS1 remains sensitive to IMP even in the presence of saturating GTP. Hence, AdSS1 has an alternative mechanism that allows IMP to inhibit in the presence of GTP (EBA2 complex of Scheme I). As will be discussed below, the alternative inhibitory site for IMP in AdSS1 may be the symmetry-related IMP pocket of the dimer.
AMP inhibits AdSS2 ~12-fold more strongly than AdSS1, and by a
different kinetic mechanism (competitive versus
noncompetitive with respect to IMP). Furthermore, in crystalline
complexes of the E. coli synthetase (52), AdSS1 (45), and
AdSS23 AMP binds only to the IMP pocket. AMP ligation of
the IMP pocket accounts for competitive inhibition of AdSS2, but
noncompetitive inhibition of AdSS1 suggests one of two alternative
mechanisms. (i) AMP promotes the dissociation of an active AdSS1 dimer
into inactive monomers. (ii) AMP inhibits one subunit of the AdSS1 dimer by binding to the IMP pocket of the other subunit. The former mechanism is unlikely, given the stabilizing effect of IMP on the dimer
(34), but the latter is plausible because of the proximity of IMP
pockets in the dimer (Fig. 5). Helix 5 and the IMP loop (the latter binds the 5'-phosphoryl group of AMP) are
conformationally dynamic elements in AdSS1 and could interact across
the subunit interface. In contrast, helix 5 in AdSS2 is immobilized by
hydrogen bond interactions and hence is less likely to transmit
conformational changes between active sites.3 IMP and AMP
inhibition in AdSS1 then could stem from a common mechanism that
involves the interaction of active sites of the dimer. AdSS2 on the
other hand seems more like the E. coli synthetase in its
properties of IMP and AMP inhibition. Kang et al. (32) have
demonstrated functionally independent (non-interacting) subunits in the
dimer of the E. coli synthetase.
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Irrespective of whether subunit interactions in the dimer are the basis for noncompetitive AMP inhibition of AdSS1, an unresolved issue still remains. How does AdSS1 exclude AMP from its IMP pocket and still retain high affinity for IMP? The conformation of the pre-Switch loop of AdSS1 in its ligand-free state differs from that of other synthetases (28), including AdSS2.3 The pre-Switch loop in AdSS1 blocks the 5'-phosphoryl pocket and holds the side chain of Asn256 in an intramolecular hydrogen bond. Hence, IMP and AMP must sacrifice binding energy to overcome the antagonistic conformation of the pre-Switch loop in AdSS1. The 4-5-fold increase in the Km of IMP and the 11-12-fold increase in Ki of AMP are consistent with less favorable interactions of each nucleotide with the IMP pocket of AdSS1 relative to AdSS2. Unlike AMP, however, IMP can in principle recover free energy lost in its binding interaction by forming 6-phosphoryl-IMP; atoms N-7 and N-6 of AMP cannot both hydrogen bond with the side chain of Asn256, whereas atoms N-7 and O-6 of 6-phosphoryl-IMP can and do hydrogen-bond with the side chain of Asn256 (44, 45). As IMP and AMP levels in most tissues are ~60 and 200 µM, respectively (53), fluctuations in the relative concentrations of AMP and IMP would influence the activity of AdSS2, whereas variations in the concentration of IMP alone would influence the activity of AdSS1.
The assignment of AdSS1 and AdSS2 to separate metabolic roles by
Nakagawa and co-workers (7, 12) rests largely on different susceptibilities to Fru-1,6-P2 inhibition (7, 12); however, the reported Ki values (0.6 and 1.6 mM
for AdSS1 and AdSS2, respectively) are 30-80-fold higher than
physiological concentrations of Fru-1,6-P2. Nonetheless,
all kinetic parameters reported by Nakagawa and co-workers (7, 10, 12,
30) are much higher than those determined by other groups (2, 6). Stayton et al. (6), for instance, report noncompetitive
Fru-1,6-P2 inhibition of AdSS1 from rat
(Ki ~130 µM) and rabbit (Ki ~50100 µM), consistent with
the findings here. Although an inhibitory site distinct from the active
site accounts for the kinetic mechanism of inhibition, a preliminary
crystallographic study reveals Fru-1,6-P2 bound as an
analogue of 6-phosphoryl-IMP.3 Fru-1,6-P2 could
be a potent competitive inhibitor with respect to IMP at low
concentrations of IMP (Ki of 16-20
µM), but when IMP is at saturation,
Fru-1,6-P2 must bind elsewhere. The location of this
alternative Fru-1,6-P2 site is unknown. The potent mode of
Fru-1,6-P2 inhibition is nearly equal for AdSS1 and AdSS2.
On the other hand, Fru-1,6-P2 binds to the alternative site
of AdSS1 with higher affinity than to that of AdSS2.
As the concentration of Fru-1,6-P2 in most tissues is ~20 µM, but can increase 3-fold in muscle during contraction (54, 55), Fru-1,6-P2 could be a dynamic regulator of AdSS1 in muscle. AdSS1 is the major if not sole form of the synthetase in muscle. The documented 60- and 3-fold increases in the in vivo concentrations of IMP and Fru-1,6-P2 during vigorous exercise should severely limit AdSS1 activity, suggesting an asynchronous PNC. Declining ATP levels in exercising muscle activate AMP deaminase, whereas the concomitant increase in levels of IMP and Fru-1,6-P2 inhibit AdSS1. Cessation of vigorous exercise leads to the restoration of ATP and Fru-1,6-P2 levels, the inactivation of AMP deaminase, and the relief of Fru-1,6-P2 inhibition of AdSS1. As the IMP concentration diminishes, the activity of AdSS1 actually accelerates until IMP is no longer saturating.
Continuous operation of the PNC in muscle is difficult to reconcile
with the accumulation of IMP in vivo and the requirement for
GTP to drive the AdSS1 reaction. The re-conversion of GDP to GTP is at
best unfavorable in the face of diminished concentrations of ATP (24).
Hence, the PNC is an unlikely source for the ammonia produced during
muscle contraction and probably cannot provide fumarate to the Krebs
cycle as originally proposed (5, 13). Indeed, partial and complete
deficiency in muscle AMP deaminase does not influence Krebs cycle
anaplerosis, phosphocreatine hydrolysis, adenine nucleotide ratios, or
exercise performance (21). Moreover, the fate of IMP accumulated during
contraction varies with muscle type (56-59); IMP primarily goes to AMP
via the PNC, but some is released as inosine and hypoxanthine. These
lost nucleotides must be re-synthesized de novo in order to
restore adenine nucleotide pools (60). Hence, AdSS1 activity related to
de novo purine biosynthesis and the PNC may be inseparable
in muscle tissue.
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FOOTNOTES |
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* This work was supported in part by National Institutes of Health Research Grant NS 10546 and National Science Foundation Grant MCB-9985565.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Dept. of Biochemistry,
Biophysics, and Molecular Biology, Molecular Biology Bldg., Iowa State
University, Ames, IA 5011. Tel.: 515-294-7103; Fax: 515-294-0453;
E-mail: hjfromm@iastate.edu.
Published, JBC Papers in Press, December 12, 2002, DOI 10.1074/jbc.M210838200
2 T. Borza and H. J. Fromm, unpublished results.
3 C. V. Iancu and R. B. Honzatko, unpublished results.
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ABBREVIATIONS |
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The abbreviations used are: PNC, purine nucleotide cycle; AdSS1, basic isozyme of mouse adenylosuccinate synthetase; AdSS2, acidic isozyme of mouse adenylosuccinate synthetase; AdSS1-Tr, N-terminal sequence truncated form of the basic isozyme; AdSS2-Tr, N-terminal sequence truncated form of the acidic isozyme; Fru-1, 6-P2, fructose 1,6-bisphosphate; PIPES, 1,4-piperazinediethanesulfonic acid; MES, 4-morpholineethanesulfonic acid; MOPS, 4-morpholinepropanesulfonic acid.
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