Kinetic and functional analysis of L-threonine kinase, the PduX enzyme of Salmonella enterica.

The PduX enzyme of Salmonella enterica is an l-threonine kinase used for the de novo synthesis of coenzyme B(12) and the assimilation of cobyric acid. PduX with an N-terminal histidine tag (His(8)-PduX) was produced in Escherichiacoli and purified. The recombinant enzyme was soluble and active. Kinetic analysis indicated a steady-state Ordered Bi Bi complex mechanism in which ATP is the first substrate to bind. Based on a multiple sequence alignment of PduX homologues and other GHMP (galactokinase, homoserine kinase, mevalonate kinase, and phosphomevalonate kinase) family members, 14 PduX variants having changes at 10 conserved serine/threonine and aspartate/glutamate sites were constructed by site-directed mutagenesis. Each variant was produced in E. coli and purified. Comparison of the circular dichroism spectra and kinetic properties of the PduX variants with those of the wild-type enzyme indicated that Glu-24 and Asp-135 are needed for proper folding, Ser-99 and Glu-132 are used for ATP binding, and Ser-253 and Ser-255 are critical to l-threonine binding whereas Ser-100 is essential to catalysis, but its precise role is uncertain. The studies reported here are the first to investigate the kinetic and catalytic mechanisms of l-threonine kinase from any organism.

Structures of mammalian and bacterial fructose-1,6-bisphosphatase reveal the basis for synergism in AMP/fructose 2,6-bisphosphate inhibition.

Fructose-1,6-bisphosphatase (FBPase) operates at a control point in mammalian gluconeogenesis, being inhibited synergistically by fructose 2,6-bisphosphate (Fru-2,6-P(2)) and AMP. AMP and Fru-2,6-P(2) bind to allosteric and active sites, respectively, but the mechanism responsible for AMP/Fru-2,6-P(2) synergy is unclear. Demonstrated here for the first time is a global conformational change in porcine FBPase induced by Fru-2,6-P(2) in the absence of AMP. The Fru-2,6-P(2) complex exhibits a subunit pair rotation of 13 degrees from the R-state (compared with the 15 degrees rotation of the T-state AMP complex) with active site loops in the disengaged conformation. A three-state thermodynamic model in which Fru-2,6-P(2) drives a conformational change to a T-like intermediate state can account for AMP/Fru-2,6-P(2) synergism in mammalian FBPases. AMP and Fru-2,6-P(2) are not synergistic inhibitors of the Type I FBPase from Escherichia coli, and consistent with that model, the complex of E. coli FBPase with Fru-2,6-P(2) remains in the R-state with dynamic loops in the engaged conformation. Evidently in porcine FBPase, the actions of AMP at the allosteric site and Fru-2,6-P(2) at the active site displace engaged dynamic loops by distinct mechanisms, resulting in similar quaternary end-states. Conceivably, Type I FBPases from all eukaryotes may undergo similar global conformational changes in response to Fru-2,6-P(2) ligation.

Structure of inhibited fructose-1,6-bisphosphatase from Escherichia coli: distinct allosteric inhibition sites for AMP and glucose 6-phosphate and the characterization of a gluconeogenic switch.

Allosteric activation of fructose-1,6-bisphosphatase (FBPase) from Escherichia coli by phosphoenolpyruvate implies rapid feed-forward activation of gluconeogenesis in heterotrophic bacteria. But how do such bacteria rapidly down-regulate an activated FBPase in order to avoid futile cycling? Demonstrated here is the allosteric inhibition of E. coli FBPase by glucose 6-phosphate (Glc-6-P), the first metabolite produced upon glucose transport into the cell. FBPase undergoes a quaternary transition from the canonical R-state to a T-like state in response to Glc-6-P and AMP ligation. By displacing Phe(15), AMP binds to an allosteric site comparable with that of mammalian FBPase. Relative movements in helices H1 and H2 perturb allosteric activator sites for phosphoenolpyruvate. Glc-6-P binds to allosteric sites heretofore not observed in previous structures, perturbing subunits that in pairs form complete active sites of FBPase. Glc-6-P and AMP are synergistic inhibitors of E. coli FBPase, placing AMP/Glc-6-P inhibition in bacteria as a possible evolutionary predecessor to AMP/fructose 2,6-bisphosphate inhibition in mammalian FBPases. With no exceptions, signature residues of allosteric activation appear in bacterial sequences along with key residues of the Glc-6-P site. FBPases in such organisms may be components of metabolic switches that allow rapid changeover between gluconeogenesis and glycolysis in response to nutrient availability.

Structures of activated fructose-1,6-bisphosphatase from Escherichia coli. Coordinate regulation of bacterial metabolism and the conservation of the R-state.

The enteric bacterium Escherichia coli requires fructose-1,6-bisphosphatase (FBPase) for growth on gluconeogenic carbon sources. Constitutive expression of FBPase and fructose-6-phosphate-1-kinase coupled with the absence of futile cycling implies an undetermined mechanism of coordinate regulation involving both enzymes. Tricarboxylic acids and phosphorylated three-carbon carboxylic acids, all intermediates of glycolysis and the tricarboxylic acid cycle, are shown here to activate E. coli FBPase. The two most potent activators, phosphoenolpyruvate and citrate, bind to the sulfate anion site, revealed previously in the first crystal structure of the E. coli enzyme. Tetramers ligated with either phosphoenolpyruvate or citrate, in contrast to the sulfate-bound structure, are in the canonical R-state of porcine FBPase but nevertheless retain sterically blocked AMP pockets. At physiologically relevant concentrations, phosphoenolpyruvate and citrate stabilize an active tetramer over a less active enzyme form of mass comparable with that of a dimer. The above implies the conservation of the R-state through evolution. FBPases of heterotrophic organisms of distantly related phylogenetic groups retain residues of the allosteric activator site and in those instances where data are available exhibit activation by phosphoenolpyruvate. Findings here unify disparate observations regarding bacterial FBPases, implicating a mechanism of feed-forward activation in bacterial central metabolism.

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.

Nucleotide complexes of Escherichia coli phosphoribosylaminoimidazole succinocarboxamide synthetase.

Phosphoribosylaminoimidazole-succinocarboxamide synthetase (SAICAR synthetase) converts 4-carboxy-5-aminoimidazole ribonucleotide (CAIR) to 4-(N-succinylcarboxamide)-5-aminoimidazole ribonucleotide (SAICAR). The enzyme is a target of natural products that impair cell growth. Reported here are the crystal structures of the ADP and the ADP.CAIR complexes of SAICAR synthetase from Escherichia coli, the latter being the first instance of a CAIR-ligated SAICAR synthetase. ADP and CAIR bind to the active site in association with three Mg(2+), two of which coordinate the same oxygen atom of the 4-carboxyl group of CAIR; whereas, the third coordinates the alpha- and beta-phosphoryl groups of ADP. The ADP.CAIR complex is the basis for a transition state model of a phosphoryl transfer reaction involving CAIR and ATP, but also supports an alternative chemical pathway in which the nucleophilic attack of l-aspartate precedes the phosphoryl transfer reaction. The polypeptide fold for residues 204-221 of the E. coli structure differs significantly from those of the ligand-free SAICAR synthetase from Thermatoga maritima and the adenine nucleotide complexes of the synthetase from Saccharomyces cerevisiae. Conformational differences between the E. coli, T. maritima, and yeast synthetases suggest the possibility of selective inhibition of de novo purine nucleotide biosynthesis in microbial organisms.

Novel allosteric activation site in Escherichia coli fructose-1,6-bisphosphatase.

Fructose-1,6-bisphosphatase (FBPase) governs a key step in gluconeogenesis, the conversion of fructose 1,6-bisphosphate into fructose 6-phosphate. In mammals, the enzyme is subject to metabolic regulation, but regulatory mechanisms of bacterial FBPases are not well understood. Presented here is the crystal structure (resolution, 1.45A) of recombinant FBPase from Escherichia coli, the first structure of a prokaryotic Type I FBPase. The E. coli enzyme is a homotetramer, but in a quaternary state between the canonical R- and T-states of porcine FBPase. Phe(15) and residues at the C-terminal side of the first alpha-helix (helix H1) occupy the AMP binding pocket. Residues at the N-terminal side of helix H1 hydrogen bond with sulfate ions buried at a subunit interface, which in porcine FBPase undergoes significant conformational change in response to allosteric effectors. Phosphoenolpyruvate and sulfate activate E. coli FBPase by at least 300%. Key residues that bind sulfate anions are conserved among many heterotrophic bacteria, but are absent in FBPases of organisms that employ fructose 2,6-bisphosphate as a regulator. These observations suggest a new mechanism of regulation in the FBPase enzyme family: anionic ligands, most likely phosphoenolpyruvate, bind to allosteric activator sites, which in turn stabilize a tetramer and a polypeptide fold that obstructs AMP binding.

Glucose 6-phosphate release of wild-type and mutant human brain hexokinases from mitochondria.

One molecule of glucose 6-phosphate inhibits brain hexokinase (HKI) with high affinity by binding to either one of two sites located in distinct halves of the enzyme. In addition to potent inhibition, glucose 6-phosphate releases HKI from the outer leaflet of mitochondria; however, the site of glucose 6-phosphate association responsible for the release of HKI is unclear. The incorporation of a C-terminal polyhistidine tag on HKI facilitates the rapid purification of recombinant enzyme from Escherichia coli. The tagged construct has N-formyl methionine as its first residue and has mitochondrial association properties comparable with native brain hexokinases. Release of wild-type and mutant hexokinases from mitochondria by glucose 6-phosphate follow equilibrium models, which explain the release phenomenon as the repartitioning of ligand-bound HKI between solution and the membrane. Mutations that block the binding of glucose 6-phosphate to the C-terminal half of HKI have little or no effect on the glucose 6-phosphate release. In contrast, mutations that block glucose 6-phosphate binding to the N-terminal half require approximately 7-fold higher concentrations of glucose 6-phosphate for the release of HKI. Results here implicate a primary role for the glucose 6-phosphate binding site at the N-terminal half of HKI in the release mechanism.

R-state AMP complex reveals initial steps of the quaternary transition of fructose-1,6-bisphosphatase.

AMP transforms fructose-1,6-bisphosphatase from its active R-state to its inactive T-state; however, the mechanism of that transformation is poorly understood. The mutation of Ala(54) to leucine destabilizes the T-state of fructose-1,6-bisphosphatase. The mutant enzyme retains wild-type levels of activity, but the concentration of AMP that causes 50% inhibition increases 50-fold. In the absence of AMP, the Leu(54) enzyme adopts an R-state conformation nearly identical to that of the wild-type enzyme. The mutant enzyme, however, grows in two crystal forms in the presence of saturating AMP. In one form, the AMP-bound tetramer is in a T-like conformation, whereas in the other form, the AMP-bound tetramer is in a R-like conformation. The latter reveals conformational changes in two helices due to the binding of AMP. Helix H1 moves toward the center of the tetramer and displaces Ile(10) from a hydrophobic pocket. The displacement of Ile(10) exposes a hydrophobic surface critical to interactions that stabilize the T-state. Helix H2 moves away from the center of the tetramer, breaking hydrogen bonds with a buried loop (residues 187-195) in an adjacent subunit. The same hydrogen bonds reform but only after the quaternary transition to the T-state. Proposed here is a model that accounts for the quaternary transition and cooperativity in the inhibition of catalysis by AMP.

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.

Origin of cooperativity in the activation of fructose-1,6-bisphosphatase by Mg2+.

Fructose-1,6-bisphosphatase requires a divalent metal cation for catalysis, Mg(2+) being its most studied activator. Phosphatase activity increases sigmoidally with the concentration of Mg(2+), but the mechanistic basis for such cooperativity is unknown. Bound magnesium cations can interact within a single subunit or between different subunits of the enzyme tetramer. Mutations of Asp(118), Asp(121), or Glu(97) to alanine inactivate the recombinant porcine enzyme. These residues bind directly to magnesium cations at the active site. Three different hybrid tetramers of fructose-1,6-bisphosphatase, composed of one wild-type subunit and three subunits bearing one of the mutations above, exhibit kinetic parameters (K(m) for fructose-1,6-bisphosphate, 1.1-1.8 microm; K(a) for Mg(2+), 0.34-0.76 mm; K(i) for fructose-2,6-bisphosphate, 0.11-0.61 microm; and IC(50) for AMP, 3.8-7.4 microm) nearly identical to those of the wild-type enzyme. Notwithstanding these similarities, the k(cat) parameter for each hybrid tetramer is approximately one-fourth of that for the wild-type enzyme. Evidently, each subunit in the wild-type tetramer can independently achieve maximum velocity when activated by Mg(2+). Moreover, the activities of the three hybrid tetramers vary sigmoidally with the concentration of Mg(2+) (Hill coefficients of approximately 2). The findings above are fully consistent with a mechanism of cooperativity that arises from within a single subunit of fructose-1,6-bisphosphatase.

Inhibition of fructose-1,6-bisphosphatase by a new class of allosteric effectors.

A highly constrained pseudo-tetrapeptide (OC252-324) further defines a new allosteric binding site located near the center of fructose-1,6-bisphosphatase. In a crystal structure, pairs of inhibitory molecules bind to opposite faces of the enzyme tetramer. Each ligand molecule is in contact with three of four subunits of the tetramer, hydrogen bonding with the side chain of Asp187 and the backbone carbonyl of residue 71, and electrostatically interacting with the backbone carbonyl of residue 51. The ligated complex adopts a quaternary structure between the canonical R- and T-states of fructose-1,6-bisphosphatase, and yet a dynamic loop essential for catalysis (residues 52-72) is in a conformation identical to that of the T-state enzyme. Inhibition by the pseudo-tetrapeptide is cooperative (Hill coefficient of 2), synergistic with both AMP and fructose 2,6-bisphosphate, noncompetitive with respect to Mg2+, and uncompetitive with respect to fructose 1,6-bisphosphate. The ligand dramatically lowers the concentration at which substrate inhibition dominates the kinetics of fructose-1,6-bisphosphatase. Elevated substrate concentrations employed in kinetic screens may have facilitated the discovery of this uncompetitive inhibitor. Moreover, the inhibitor could mimic an unknown natural effector of fructose-1,6-bisphosphatase, as it interacts strongly with a conserved residue of undetermined functional significance.

Metaphosphate in the active site of fructose-1,6-bisphosphatase.

The hydrolysis of a phosphate ester can proceed through an intermediate of metaphosphate (dissociative mechanism) or through a trigonal bipryamidal transition state (associative mechanism). Model systems in solution support the dissociative pathway, whereas most enzymologists favor an associative mechanism for enzyme-catalyzed reactions. Crystals of fructose-1,6-bisphosphatase grow from an equilibrium mixture of substrates and products at near atomic resolution (1.3 A). At neutral pH, products of the reaction (orthophosphate and fructose 6-phosphate) bind to the active site in a manner consistent with an associative reaction pathway; however, in the presence of inhibitory concentrations of K+ (200 mm), or at pH 9.6, metaphosphate and water (or OH-) are in equilibrium with orthophosphate. Furthermore, one of the magnesium cations in the pH 9.6 complex resides in an alternative position, and suggests the possibility of metal cation migration as the 1-phosphoryl group of the substrate undergoes hydrolysis. To the best of our knowledge, the crystal structures reported here represent the first direct observation of metaphosphate in a condensed phase and may provide the structural basis for fundamental changes in the catalytic mechanism of fructose-1,6-bisphosphatase in response to pH and different metal cation activators.

Interaction of Tl+ with product complexes of fructose-1,6-bisphosphatase.

Fructose-1,6-bisphosphatase requires divalent cations (Mg2+, Mn2+, or Zn2+) for catalysis, but a diverse set of monovalent cations (K+, Tl+, Rb+, or NH(4)(+)) will further enhance enzyme activity. Here, the interaction of Tl+ with fructose-1,6-bisphosphatase is explored under conditions that support catalysis. On the basis of initial velocity kinetics, Tl+ enhances catalysis by 20% with a K(a) of 1.3 mm and a Hill coefficient near unity. Crystal structures of enzyme complexes with Mg2+, Tl+, and reaction products, in which the concentration of Tl+ is 1 mm or less, reveal Mg2+ at metal sites 1, 2, and 3 of the active site, but little or no bound Tl+. Intermediate concentrations of Tl+ (5-20 mm) displace Mg2+ from site 3 and the 1-OH group of fructose 6-phosphate from in-line geometry with respect to bound orthophosphate. Loop 52-72 appears in a new conformational state, differing from its engaged conformation by disorder in residues 61-69. Tl+ does not bind to metal sites 1 or 2 in the presence of Mg2+, but does bind to four other sites with partial occupancy. Two of four Tl+ sites probably represent alternative binding sites for the site 3 catalytic Mg2+, whereas the other sites could play roles in monovalent cation activation.

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.

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.

IMP, GTP, and 6-phosphoryl-IMP complexes of recombinant mouse muscle adenylosuccinate synthetase.

Prokaryotes have a single form of adenylosuccinate synthetase that controls the committed step of AMP biosynthesis, but vertebrates have two isozymes of the synthetase. The basic isozyme, which predominates in muscle, participates in the purine nucleotide cycle, has an active site conformation different from that of the Escherichia coli enzyme, and exhibits significant differences in ligand recognition. Crystalline complexes presented here of the recombinant basic isozyme from mouse show the following. GTP alone binds to the active site without inducing a conformational change. IMP in combination with an acetate anion induces major conformational changes and organizes the active site for catalysis. IMP, in the absence of GTP, binds to the GTP pocket of the synthetase. The combination of GTP and IMP results in the formation of a stable complex of 6-phosphoryl-IMP and GDP in the presence or absence of hadacidin. The response of the basic isozyme to GTP alone differs from that of synthetases from plants, and yet the conformation of the mouse basic and E. coli synthetases in their complexes with GDP, 6-phosphoryl-IMP, and hadacidin are nearly identical. Hence, reported differences in ligand recognition among synthetases probably arise from conformational variations observed in partially ligated enzymes.

Hybrid tetramers of porcine liver fructose-1,6-bisphosphatase reveal multiple pathways of allosteric inhibition.

Fructose-1,6-bisphosphatase is a square planar tetramer of identical subunits, which exhibits cooperative allosteric inhibition of catalysis by AMP. Protocols for in vitro subunit exchange provide three of five possible hybrid tetramers of fructose-1,6-bisphosphatase in high purity. The two hybrid types with different subunits in the top and bottom halves of the tetramer co-purify. Hybrid tetramers, formed from subunits unable to bind AMP and subunits with wild-type properties, differ from the wild-type enzyme only in regard to their properties of AMP inhibition. Hybrid tetramers exhibit cooperative, potent, and complete (100%) AMP inhibition if at least one functional AMP binding site exists in the top and bottom halves of the tetramer. Furthermore, titrations of hybrid tetramers with AMP, monitored by a tryptophan reporter group, reveal cooperativity and fluorescence changes consistent with an R- to T-state transition, provided that again at least one functional AMP site exists in the top and bottom halves of the tetramer. In contrast, hybrid tetramers, which have functional AMP binding sites in only one half (top/bottom), exhibit an R- to T-state transition and complete AMP inhibition, but without cooperativity. Evidently, two pathways of allosteric inhibition of fructose-1,6-bisphosphatase are possible, only one of which is cooperative.

Determinants of L-aspartate and IMP recognition in Escherichia coli adenylosuccinate synthetase.

Adenylosuccinate synthetase governs the first committed step in the de novo synthesis of AMP. Mutations of conserved residues in the synthetase from Escherichia coli reveal significant roles for Val(273) and Thr(300) in the recognition of l-aspartate, even though these residues do not or cannot hydrogen bond with the substrate. The mutation of Thr(300) to alanine increases the K(m) for l-aspartate by 30-fold. In contrast, its mutation to valine causes no more than a 4-fold increase in the K(m) for l-aspartate, while increasing k(cat) by 3-fold. Mutations of Val(273) to alanine, threonine, or asparagine increase the K(m) for l-aspartate from 15- to 40-fold, and concomitantly decrease the K(i) for dicarboxylate analogues of l-aspartate by up to 40-fold. The above perturbations are comparable with those resulting from the elimination of a hydrogen bond between the enzyme and substrate: alanine mutations of Thr(128) and Thr(129) increase the K(m) for IMP by up to 30-fold and the alanine mutation of Thr(301) abolishes catalysis supported by l-aspartate, but has no effect on catalysis supported by hydroxylamine. Structure-based mechanisms, by which the above residues influence substrate recognition, are presented.

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.