Mechanistic implications from crystalline complexes of wild-type and mutant adenylosuccinate synthetases from Escherichia coli.

Asp13 and His41 are essential residues of adenylosuccinate synthetase, putatively catalyzing the formation of adenylosuccinate from an intermediate of 6-phosphoryl-IMP. Wild-type adenylosuccinate synthetase and three mutant synthetases (Arg143 --> Leu, Lys16 --> Gln, and Arg303 --> Leu) from Eschericha coli have been crystallized in the presence of IMP, hadacidin (an analogue of L-aspartate), Mg2+, and GTP. The active site of each complex contains 6-phosphoryl-IMP, Mg2+, GDP, and hadacidin, except for the Arg303 --> Leu mutant, which does not bind hadacidin. In response to the formation of 6-phosphoryl-IMP, Asp13 enters the inner coordination sphere of the active site Mg2+. His41 hydrogen bonds with 6-phosphoryl-IMP, except in the Arg303 --> Leu complex, where it remains bound to the guanine nucleotide. Hence, recognition of the active site Mg2+ by Asp13 evidently occurs after the formation of 6-phosphoryl-IMP, but recognition of the intermediate by His41 may require the association of L-aspartate with the active site. Structures reported here support a mechanism in which Asp13 and His41 act as the catalytic base and acid, respectively, in the formation of 6-phosphoryl-IMP, and then act together as catalytic acids in the subsequent formation of adenylosuccinate.     

Recombinant mouse muscle adenylosuccinate synthetase: overexpression, kinetics, and crystal structure

Vertebrates possess two isozymes of adenylosuccinate synthetase. The acidic isozyme is similar to the synthetase from bacteria and plants, being involved in the de novo biosynthesis of AMP, whereas the basic isozyme participates in the purine nucleotide cycle. Reported here is the first instance of overexpression and crystal structure determination of a basic isozyme of adenylosuccinate synthetase. The recombinant mouse muscle enzyme purified to homogeneity in milligram quantities exhibits a specific activity comparable with that of the rat muscle enzyme isolated from tissue and K(m) parameters for GTP, IMP, and l-aspartate (12, 45, and 140 microm, respectively) similar to those of the enzyme from Escherichia coli. The mouse muscle and E. coli enzymes have similar polypeptide folds, differing primarily in the conformation of loops, involved in substrate recognition and stabilization of the transition state. Residues 65-68 of the muscle isozyme adopt a conformation not observed in any previous synthetase structure. In its new conformation, segment 65-68 forms intramolecular hydrogen bonds with residues essential for the recognition of IMP and, in fact, sterically excludes IMP from the active site. Observed differences in ligand recognition among adenylosuccinate synthetases may be due in part to conformational variations in the IMP pocket of the ligand-free enzymes.     

Cavitation as a mechanism of substrate discrimination by adenylosuccinate synthetases

Adenylosuccinate synthetase catalyzes the first committed step in the de novo biosynthesis of AMP, coupling L-aspartate and IMP to form adenylosuccinate. Km values of IMP and 2'-deoxy-IMP are nearly identical with each substrate supporting comparable maximal velocities. Nonetheless, the Km value for L-aspartate and the Ki value for hadacidin (a competitive inhibitor with respect to L-aspartate) are 29-57-fold lower in the presence of IMP than in the presence of 2'-deoxy-IMP. Crystal structures of the synthetase ligated with hadacidin, GDP, and either 6-phosphoryl-IMP or 2'-deoxy-6-phosphoryl-IMP are identical except for the presence of a cavity normally occupied by the 2'-hydroxyl group of IMP. In the presence of 6-phosphoryl-IMP and GDP (hadacidin absent), the L-aspartate pocket can retain its fully ligated conformation, forming hydrogen bonds between the 2'-hydroxyl group of IMP and sequence-invariant residues. In the presence of 2'-deoxy-6-phosphoryl-IMP and GDP, however, the L-aspartate pocket is poorly ordered. The absence of the 2'-hydroxyl group of the deoxyribonucleotide may destabilize binding of the ligand to the L-aspartate pocket by disrupting hydrogen bonds that maintain a favorable protein conformation and by the introduction of a cavity into the fully ligated active site. At an approximate energy cost of 2.2 kcal/mol, the unfavorable thermodynamics of cavity formation may be the major factor in destabilizing ligands at the L-aspartate pocket.     

Feedback inhibition and product complexes of recombinant mouse muscle adenylosuccinate synthetase

Adenylosuccinate synthetase governs the committed step of AMP biosynthesis, the generation of 6-phosphoryl-IMP from GTP and IMP followed by the formation of adenylosuccinate from 6-phosphoryl-IMP and l-aspartate. The enzyme is subject to feedback inhibition by AMP and adenylosuccinate, but crystallographic complexes of the mouse muscle synthetase presented here infer mechanisms of inhibition that involve potentially synergistic ligand combinations. AMP alone adopts the productive binding mode of IMP and yet stabilizes the active site in a conformation that favors the binding of Mg(2+)-IMP to the GTP pocket. On the other hand, AMP, in the presence of GDP, orthophosphate, and Mg(2+), adopts the binding mode of adenylosuccinate. Depending on circumstances then, AMP behaves as an analogue of IMP or as an analogue of adenylosuccinate. The complex of adenylosuccinate.GDP.Mg(2+).sulfate, the first structure of an adenylosuccinate-bound synthetase, reveals significant geometric distortions and tight nonbonded contacts relevant to the proposed catalytic mechanism. Adenylosuccinate forms from 6-phosphoryl-IMP and l-aspartate by the movement of the purine ring into the alpha-amino group of l-aspartate.     

Effectors of the stringent response target the active site of Escherichia coli adenylosuccinate synthetase.

Guanosine 5'-diphosphate 3'-diphosphate (ppGpp), a pleiotropic effector of the stringent response, potently inhibits adenylosuccinate synthetase from Escherichia coli as an allosteric effector and/or as a competitive inhibitor with respect to GTP. Crystals of the synthetase grown in the presence of IMP, hadacidin, NO3-, and Mg2+, then soaked with ppGpp, reveal electron density at the GTP pocket which is consistent with guanosine 5'-diphosphate 2':3'-cyclic monophosphate. Unlike ligand complexes of the synthetase involving IMP and GDP, the coordination of Mg2+ in this complex is octahedral with the side chain of Asp13 in the inner sphere of the cation. The cyclic phosphoryl group interacts directly with the side chain of Lys49 and indirectly through bridging water molecules with the side chains of Asn295 and Arg305. The synthetase either directly facilitates the formation of the cyclic nucleotide or scavenges trace amounts of the cyclic nucleotide from solution. Regardless of its mode of generation, the cyclic nucleotide binds far more tightly to the active site than does ppGpp. Conceivably, synthetase activity in vivo during the stringent response may be sensitive to the relative concentrations of several effectors, which together exercise precise control over the de novo synthesis of AMP.     

IMP Alone Organizes the Active Site of Adenylosuccinate Synthetase from Escherichia coli.

A complete set of substrate/substrate analogs of adenylosuccinate synthetase from Escherichia coli induces dimer formation and a transition from a disordered to an ordered active site. The most striking of the ligand-induced effects is the movement of loop 40-53 by up to 9 A. Crystal structures of the partially ligated synthetase, which either combine IMP and hadacidin or IMP, hadacidin, and Mg(2+)-pyrophosphate, have ordered active sites, comparable with the fully ligated enzyme. More significantly, a crystal structure of the synthetase with IMP alone exhibits a largely ordered active site, which includes the 9 A movement of loop 40-53 but does not include conformational adjustments to backbone carbonyl 40 (Mg(2+) interaction element) and loop 298-304 (L-aspartate binding element). Interactions involving the 5'-phosphoryl group of IMP evidently trigger the formation of salt links some 30 A away. The above provides a structural basis for ligand binding synergism, effects on k(cat) due to mutations far from the site of catalysis, and the complete loss of substrate efficacy due to minor alterations of the 5'-phosphoryl group of IMP.     

Studies of ligand binding to Escherichia coli adenylosuccinate synthetase

Dissociation constants of Escherichia coli adenylosuccinate synthetase with IMP, GTP, adenylosuccinate, and AMP (a competitive inhibitor for IMP) were determined by measuring the extent of quenching of the intrinsic tryptophan fluorescence of the enzyme. The enzyme has one binding site for each of these ligands. Aspartate and GDP did not quench the fluorescence to any great extent, and their dissociation constants could not be determined. These ligand binding studies were generally supportive of the kinetic mechanism proposed earlier for the enzyme. Cys291 was modified with the fluorescent chromophores N-(iodoacetylaminoethyl)-5-naphthylamine-1-sulfonate and tetramethylrhodamine maleimide in order to measure enzyme conformational changes attending ligand binding. The excitation and emission spectra of these fluorophores are not altered by the addition of active site binding ligands. TbGTP and TbGDP were used as native reporter groups, and changes in their fluorescence on complexing with the enzyme and various ligands made it possible to detect conformational changes occurring at the active site. Evidence is presented for abortive complexes of the type: enzyme-TbGTP-adenylosuccinate and enzyme-TbGTP-adenylosuccinate-aspartate. These results suggest that the IMP and aspartate binding sites are spatially separated.     

Variations in the response of mouse isozymes of adenylosuccinate synthetase to inhibitors of physiological relevance

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.     

An intermediate step in the recognition of tRNA(Asp) by aspartyl-tRNA synthetase

The crystal structures of aspartyl-tRNA synthetase (AspRS) from Thermus thermophilus, a prokaryotic class IIb enzyme, complexed with tRNA(Asp) from either T. thermophilus or Escherichia coli reveal a potential intermediate of the recognition process. The tRNA is positioned on the enzyme such that it cannot be aminoacylated but adopts an overall conformation similar to that observed in active complexes. While the anticodon loop binds to the N-terminal domain of the enzyme in a manner similar to that of the related active complexes, its aminoacyl acceptor arm remains at the entrance of the active site, stabilized in its intermediate conformational state by non-specific interactions with the insertion and catalytic domains. The thermophilic nature of the enzyme, which manifests itself in a very low kinetic efficiency at 17 degrees C, the temperature at which the crystals were grown, is in agreement with the relative stability of this non-productive conformational state. Based on these data, a pathway for tRNA binding and recognition is proposed.     

Studies on active site mutants of P. falciparum adenylosuccinate synthetase: Insights into enzyme catalysis and activation

Adenylosuccinate synthetase catalyzes a reversible reaction utilizing IMP, GTP and aspartate in the presence of Mg²⁺ to form adenylosuccinate, GDP and inorganic phosphate. Comparison of similarly liganded complexes of Plasmodium falciparum, mouse and Escherichia coli AdSS reveals H-bonding interactions involving nonconserved catalytic loop residues (Asn429, Lys62 and Thr307) that are unique to the parasite enzyme. Site-directed mutagenesis has been used to examine the role of these interactions in catalysis and structural organization of P. falciparum adenylosuccinate synthetase (PfAdSS). Mutation of Asn429 to Val, Lys62 to Leu and Thr307 to Val resulted in an increase in K_m values for IMP, GTP and aspartate, respectively along with a 5 fold drop in the k_cat value for N429V mutant suggesting the role of these residues in ligand binding and/or catalysis. We have earlier shown that the glycolytic intermediate, fructose 1,6 bisphosphate, which is an inhibitor of mammalian AdSS is an activator of the parasite enzyme. Enzyme kinetics along with molecular docking suggests a mechanism for activation wherein F16BP seems to be binding to the Asp loop and inducing a conformation that facilitates aspartate binding to the enzyme active site. Like in other AdSS, a conserved arginine residue (Arg155) is involved in dimer crosstalk and interacts with IMP in the active site of the symmetry related subunit of PfAdSS. We also report on the biochemical characterization of the arginine mutants (R155L, R155K and R155A) which suggests that unlike in E. coli AdSS, Arg155 in PfAdSS influences both ligand binding and catalysis.     

Adenylosuccinate synthetase from Dictyostelium discoideum: effects of hadacidin analogs and binding of [14C]hadacidin

The enzyme adenylosuccinate (sAMP) synthetase has been partially purified from Dictyostelium discoideum using hadacidin-Sepharose 4B affinity chromatography, anion-exchange high-performance liquid chromatography (HPLC), and gel-filtration HPLC, resulting in a 2600-fold purification. Using a newly developed HPLC procedure to assay activity, it has been found that D. discoideum adenylosuccinate synthetase activity has apparent Km values for the substrates IMP, GTP, and aspartate of 36, 23, and 714 microM, respectively. The analog guanosine-5'-(beta, gamma-imino)triphosphate was found to be an inhibitor of GTP with a Ki of 15 microM, and IMP was competitively inhibited by its analog formycin B monophosphate with a Ki of 80 microM. An analysis of these kinetic data showed a pattern consistent with a fully random terter mechanism. Hadacidin, an analog of aspartate, was an inhibitor of that substrate at 86 microM. Other analogs of hadacidin were synthesized and examined for their effect on the sAMP synthetase activity. Compared to hadacidin, which produced 100% inhibition at 5 mM, it was observed that N-acetyl-N-hydroxyglycine, N-formylglycine, N-acetylglycine, and N-hydroxyglycine all inhibited between 50 and 75%, with N-(thiocarboxy)-L-aspartic anhydride less effective at 27%, and N-benzoylglycine at only 6%. N-Formylsarcosine, N-acetylmethionine, O-methylpyruvate oxime, and hadacidin methylester had no effect at this concentration. The adenylosuccinate synthetase activity was dependent on metal ions with maximum activity being obtained with Mg2+. The ability of the aspartate analog hadacidin to bind to the purified adenylosuccinate synthetase was demonstrated using anion-exchange HPLC and [formyl-14C]hadacidin. The radioactivity coeluted with the adenylosuccinate synthetase and the bound, radiolabeled hadacidin was displaced by excess aspartate.     

Conformational changes in the bacterial SRP receptor FtsY upon binding of guanine nucleotides and SRP

In cotranslational preprotein targeting in Escherichia coli, the signal recognition particle (SRP) binds to the signal peptide emerging from the ribosome and, subsequently, interacts with the signal recognition particle receptor, FtsY, at the plasma membrane. Both FtsY and the protein moiety of the signal recognition particle, Ffh, are GTPases, and GTP is required for the formation of the SRP-FtsY complex. We have studied the binding of GTP/GDP to FtsY as well as the SRP-FtsY complex formation by monitoring the fluorescence of tryptophan 343 in the I box of mutant FtsY. Thermodynamic and kinetic parameters of the FtsY complexes with GDP, GTP, and signal recognition particle are reported. Upon SRP-FtsY complex formation in the presence of GTP, the fluorescence of tryptophan 343 increased by 50 % and was blue-shifted by 10 nm. We conclude that GTP-dependent SRP-FtsY complex formation leads to an extensive conformational change in the I box insertion in the effector region of FtsY.     

The mechanism of the adenylosuccinate synthetase reaction as studied by positional isotope exchange

In an attempt to gain insight into the mechanism of the rat muscle adenylosuccinate synthetase reaction, experiments using the technique of positional isotope exchange (isotope scrambling) were undertaken. [gamma-18O]GTP was prepared and incubated with Mg2+ and the synthetase in the presence of various ligands. Positional isotope exchange occurred, as measured by nuclear magnetic resonance spectroscopy, when IMP was present. In the absence of IMP, with or without aspartate or succinate, the [gamma-18O]GTP did not exhibit scrambling. These results suggest that the adenylosuccinate synthetase reaction involves the participation of 6-phosphoryl-IMP as an obligatory intermediate. On the basis of experiments carried out in our laboratory as well as in others, we believe the GDP remains bound to the enzyme until the product, adenylosuccinate, is formed. All products may then dissociate randomly from the enzyme. The positional isotope exchange experiments, along with initial-rate experiments carried out in our laboratory, serve to explain the lack of partial exchange reactions associated with the synthetase (Fromm, H. J. (1958) Biochim. Biophys. Acta 29, 255-262), as well as the net inversion of configuration when chiral thio-GTP is converted to thiophosphate (Webb, M. R., Reed, G. H., Cooper, B. F., and Rudolph, F. B. (1984) J. Biol. Chem. 259, 3044-3046).     

Crystal structure of fully ligated adenylosuccinate synthetase from Plasmodium falciparum

In the absence of the de novo purine nucleotide biosynthetic pathway in parasitic protozoa, purine salvage is of primary importance for parasite survival. Enzymes of the salvage pathway are, therefore, good targets for anti-parasitic drugs. Adenylosuccinate synthetase (AdSS), catalysing the first committed step in the synthesis of AMP from IMP, is a potential target for anti-protozoal chemotherapy. We report here the crystal structure of adenylosuccinate synthetase from the malaria parasite, Plasmodium falciparum, complexed to 6-phosphoryl IMP, GDP, Mg2+ and the aspartate analogue, hadacidin at 2 A resolution. The overall architecture of P. falciparum AdSS (PfAdSS) is similar to the known structures from Escherichia coli, mouse and plants. Differences in substrate interactions seen in this structure provide a plausible explanation for the kinetic differences between PfAdSS and the enzyme from other species. Additional hydrogen bonding interactions of the protein with GDP may account for the ordered binding of substrates to the enzyme. The dimer interface of PfAdSS is also different, with a pronounced excess of positively charged residues. Differences highlighted here provide a basis for the design of species-specific inhibitors of the enzyme.     

Theoretical Study of the Structure of Adenosine Deaminase Complexes with Adenosine Analogues: I. Aza-, Deaza-, and Isomeric Azadeazaanalogues of Adenosine

Conformational models of the active site of adenosine deaminase (ADA) and its complexes in the basic state with adenosine and 13-isosteric analogues of the aza, deaza, and azadeaza series were constructed. The optimization of the conformational energy of the active site and the nucleoside bound with it in the complex was achieved in the force field of the whole enzyme [the structure of ADA complex with 1-deazaadenosine (1ADD) was used] within the molecular mechanics model using the AMBER 99 potentials. The stable conformational states of each of the complexes, as well as the optimal conformation of ADA in the absence of ligand, were determined. It was proved that the conformational state that is close to the structure of the ADA complex with 1ADD known from X-ray study corresponds to one of the local minima of the potential surface. Another, a significantly deeper minimum was determined; it differs from the first minimum by the mutual orientation of side chains of amino acid residues. A similar conformational state is optimal for the ADA active site in the absence of bound ligand. A qualitative correlation exists between the values of potential energies of the complexes in this conformation and the enzymatic activity of ADA toward the corresponding nucleosides. The dynamics of conformational conversions of the active site after the binding of substrate or its analogues, as well as the possibility of the estimation of the inhibitory properties of nucleosides on the basis of calculations, are discussed.     

Nuclear overhauser effect studies of the conformations of Mg(alpha, beta-methylene)ATP bound to E. coli isoleucyl-tRNA synthetase

Internuclear distances obtained from transferred nuclear Overhauser effects were used in combination with distance geometry calculations to define the E. coli isoleucyl-tRNA synthetase bound conformation of Mg(alpha, beta-methylene)ATP both in the absence and in the presence of the cognate and noncognate amino acids L-isoleucine and L-valine, respectively. A single nucleotide structure having an anti adenine-ribose glycosidic torsional angle of -114 degrees was found to satisfy the experimental distance constraints. The nearly identical anti glycosidic torsional angles observed in all three complexes demonstrate that the conformation of the adenosine moiety of the enzyme-bound nucleotide is not sensitive to the presence or to the nature of the amino acid bound at the aminoacyladenylate site. In addition, the acceptable range of Mg(alpha, beta-methylene)ATP conformations bound to the E. coli isoleucyl-tRNA synthetase was found to be nearly identical to that previously determined for the E. coli methionyl-tRNA synthetase (Williams and Rosevear (1991) J. Biol. Chem. 266, 2089-2098). Thus, the predicted structural homology between the isoleucyl- and methionyl-tRNA synthetases, both members of the same class of synthetases on the basis of common consensus sequences, is further supported by consensus enzyme-bound nucleotide conformations.     

Theoretical study of the structure of adenosine deaminase complexes with adenosine analogues: I. Aza-, deaza- and isomeric azadeazaanalogues of adenosine

The conformational models of the active site of adenosine deaminase (ADA) and its complexes in the basic state with adenosine and 13 isosteric analogues of the aza, deaza, and azadeaza series were constructed. The optimization of the conformational energy of the active site and the nucleoside bound with it in the complex was achieved in the force field of the whole enzyme (the 1ADD structure was used) within the molecular mechanics model using the AMBER 99 potentials. The stable conformational states of each of the complexes, as well as the optimal conformation of the ADA in the absence of ligand, were determined. It was proved that the conformational state that is close to the structure of the ADA complex with 1-deazaadenosine (1ADD) known from the X-ray study corresponds to one of the local minima of the potential surface. Another, a significantly deeper minimum was determined; it differs from the first minimum by the mutual orientation of side chains of amino acid residues. A similar conformational state is optimal for the ADA active site in the absence of the bound ligand. A qualitative correlation exists between the values of potential energies of the complexes in this conformation and the enzymatic activity of ADA toward the corresponding nucleosides. The dynamics of conformational conversions of the active site after the binding of substrate or its analogues, as well as the possibility of the estimation of the inhibitory properties of nucleosides on the basis of calculations, are discussed.     

Conformational selection through electrostatics: Free energy simulations of GTP and GDP binding to archaeal initiation factor 2

Archaeal Initiation Factor 2 is a GTPase involved in protein biosynthesis. In its GTP-bound, "ON" conformation, it binds an initiator tRNA and carries it to the ribosome. In its GDP-bound, "OFF" conformation, it dissociates from tRNA. To understand the specific binding of GTP and GDP and their dependence on the conformational state, molecular dynamics free energy simulations were performed. The ON state specificity was predicted to be weak, with a GTP/GDP binding free energy difference of -1 kcal/mol, favoring GTP. The OFF state specificity is larger, 4 kcal/mol, favoring GDP. The overall effects result from a competition among many interactions in several complexes. To interpret them, we use a simpler, dielectric continuum model. Several effects are robust with respect to the model details. Both nucleotides have a net negative charge, so that removing them from solvent into the binding pocket carries a desolvation penalty, which is large for the ON state, and strongly disfavors GTP binding compared to GDP. Short-range interactions between the additional GTP phosphate group and ionized sidechains in the binding pocket offset most, but not all of the desolvation penalty; more distant groups also contribute significantly, and the switch 1 loop only slightly. The desolvation penalty is lower for the more open, wetter OFF state, and the GTP/GDP difference much smaller. Short-range interactions in the binding pocket and with more distant groups again make a significant contribution. Overall, the simulations help explain how conformational selection is achieved with a single phosphate group.     

Structure of Mycobacterium tuberculosis FtsZ reveals unexpected, G protein-like conformational switches

We report three crystal structures of the Mycobacterium tuberculosis cell division protein FtsZ, as the citrate, GDP, and GTPgammaS complexes, determined at 1.89, 2.60, and 2.08A resolution. MtbFtsZ crystallized as a tight, laterally oriented dimer distinct from the longitudinal polymer observed for alphabeta-tubulin. Mutational data on Escherichia coli FtsZ suggest that this dimer interface is important for proper protofilament and "Z-ring" assembly and function. An alpha-to-beta secondary structure conformational switch at the dimer interface is spatially analogous to, and has many of the hallmarks of, the Switch I conformational changes exhibited by G-proteins upon activation. The presence of a gamma-phosphate in the FtsZ active site modulates the conformation of the "tubulin" loop T3 (spatially analogous to the G-protein Switch II); T3 switching upon gamma-phosphate ligation is directly coupled to the alpha-to-beta switch by steric overlap. The dual conformational switches observed here for the first time in an FtsZ link GTP binding and hydrolysis to FtsZ (and tubulin) lateral assembly and Z-ring contraction, and they are suggestive of an underappreciated functional analogy between FtsZ, tubulin and G-proteins.     

Mechanism of action of Escherichia coli phosphoribosylaminoimidazolesuccinocarboxamide synthetase

The conversion of ATP, L-aspartate, and 5-aminoimidazole-4-carboxyribonucleotide (CAIR) to 5-aminoimidazole-4-(N-succinylcarboxamide) ribonucleotide (SAICAR), ADP, and phosphate by phosphoribosylaminoimidazolesuccinocarboxamide synthetase (SAICAR synthetase) represents the eighth step of de novo purine nucleotide biosynthesis. SAICAR synthetase and other enzymes of purine biosynthesis are targets of natural products that impair cell growth. Prior to this study, no kinetic mechanism was known for any SAICAR synthetase. Here, a rapid equilibrium random ter-ter kinetic mechanism is established for the synthetase from Escherichia coli by initial velocity kinetics and patterns of linear inhibition by IMP, adenosine 5'-(beta,gamma-imido)triphosphate (AMP-PNP), and maleate. Substrates exhibit mutual binding antagonism, with the strongest antagonism between CAIR and either ATP or L-aspartate. CAIR binds to the free enzyme up to 200-fold more tightly than to the ternary enzyme-ATP-aspartate complex, but the latter complex may be the dominant form of SAICAR synthetase in vivo. IMP is a competitive inhibitor with respect to CAIR, suggesting the possibility of a hydrogen bond interaction between the 4-carboxyl and 5-amino groups of enzyme-bound CAIR. Of several aspartate analogues tested (hadacidin, l-malate, succinate, fumarate, and maleate), maleate was by far the best inhibitor, competitive with respect to L-aspartate. Inhibition by IMP and maleate is consistent with a chemical mechanism for SAICAR synthetase that parallels that of adenylosuccinate synthetase.