Enzyme–substrate complexes of allosteric citrate synthase: Evidence for a novel intermediate in substrate binding

The citrate synthase (CS) of Escherichia coli is an allosteric hexameric enzyme specifically inhibited by NADH. The crystal structure of wild type (WT) E. coli CS, determined by us previously, has no substrates bound, and part of the active site is in a highly mobile region that is shifted from the position needed for catalysis. The CS of Acetobacter aceti has a similar structure, but has been successfully crystallized with bound substrates: both oxaloacetic acid (OAA) and an analog of acetyl coenzyme A (AcCoA). We engineered a variant of E. coli CS wherein five amino acids in the mobile region have been replaced by those in the A. aceti sequence. The purified enzyme shows unusual kinetics with a low affinity for both substrates. Although the crystal structure without ligands is very similar to that of the WT enzyme (except in the mutated region), complexes are formed with both substrates and the allosteric inhibitor NADH. The complex with OAA in the active site identifies a novel OAA-binding residue, Arg306, which has no functional counterpart in other known CS-OAA complexes. This structure may represent an intermediate in a multi-step substrate binding process where Arg306 changes roles from OAA binding to AcCoA binding. The second complex has the substrate analog, S-carboxymethyl-coenzyme A, in the allosteric NADH-binding site and the AcCoA site is not formed. Additional CS variants unable to bind adenylates at the allosteric site show that this second complex is not a factor in positive allosteric activation of AcCoA binding.     

The binding of reduced nicotinamide adenine dinucleotide to citrate synthase of Escherichia coli K12.

Citrate synthase from Escherichia coli enhances the fluorescence of its allosteric inhibitor, NADH, and shifts the peak of emission of the coenzyme from 457 to 428 nm. These effects have been used to measure the binding of NADH to this enzyme under various conditions. The dissociation constant for the NADH-citrate synthase complex is about 0.28 muM at pH 6.2, but increases toward alkaline pH as if binding depends on protonation of a group with a pKa of about 7.05. Over the pH range 6.2-8.7, the number of binding sites decreases from about 0.65 to about 0.25 per citrate synthase subunit. The midpoint of this transition is at about pH 7.7, and it may be one reflection of the partial depolymerization of the enzyme which is known to occur in this pH range. A gel filtration method has been used to verify that the fluorescence enhancement technique accurately reveals all of the NADH molecules bound to the enzyme in the concentration range of interest. NAD+ and NADP+ were weak competitive inhibitors of NADH binding at pH 7.8 (Ki values greater than 1 mM), but stronger inhibition was shown by 5'-AMP and 3'-AMP, with Ki values of 83 +/- 5 and 65 +/- 4 muM, respectively. Acetyl-CoA, one of the substrates, and KCl, an activator, also inhibit the binding in a weakly cooperative manner. All of these effects are consistent with kinetic observations on this system. We interpret our results in terms of two types of binding site for nucleotides on citrate synthase: an active site which binds acetyl-CoA, the substrate, or its analogue 3'-AMP; and an allosteric site which binds NADH or its analogue 5'-AMP and has a lesser affinity for other nicotinamide adenine dinucloetides. When the active site is occupied, we propose that NADH cannot bind to the allosteric site, but 5'-AMP can; conversely, when NADH is the in the allosteric site, the active site cannot be occupied. In addition to these two classes of sites, there must be points for interaction with KCl and other salts. Oxaloacetate, the second substrate, and alpha-ketoglutarate, an inhibitor whose mode of action is believed to be allosteric, have no effect on NADH binding to citrate synthase at pH 7.8. When NADH is bound to citrate synthase, it quenches the intrinsic tryptophan fluorescence of the enzyme. The amount of quenching is proportional to the amount of NADH bound, at least up to a binding ratio of 0.50 NADH per enzyme subunit. This amount of binding leads to the quenching of 53 +/- 5% of the enzyme fluorescence, which means that one NADH molecule can quench all the intrinsic fluorescence of the subunit to which it binds.     

In vitro mutagenesis of Escherichia coli citrate synthase to clarify the locations of ligand binding sites

In vitro mutagenesis techniques have been used to investigate two structure-function questions relating to the allosteric citrate synthase of Escherichia coli. The first question concerns the binding site of alpha-keto-glutarate, which is a structural analogue of the substrate oxaloacetate and yet has been suggested to be an allosteric inhibitor of the enzyme. Using oligonucleotide-directed mutagenesis of the cloned E. coli citrate synthase gene, we prepared missense mutants, designated CS226H----Q and CS229H----Q, in which histidine residues at positions 226 and 229, respectively, were replaced by glutamine. In the homologous pig heart citrate synthase it is known (Wiegand, G., and Remington, S. J. (1986) Annu. Rev. Biophys. Biophys. Chem. 15, 97-117) that the equivalent of His-229 helps to bind oxaloacetate, while the equivalent of His-226 is nearby. Kinetic and ligand binding measurements showed that CS226H----Q had a reduced affinity for oxaloacetate and alpha-ketoglutarate, while CS229H----Q bound oxaloacetate even less effectively, and was not inhibited by alpha-ketoglutarate at all under our conditions. This parallel loss of binding affinities for oxaloacetate and alpha-ketoglutarate, in two mutants altered in residues at the active site of E. coli citrate synthase, strongly suggests that inhibition of this enzyme by alpha-ketoglutarate is not allosteric but occurs by competitive inhibition at the active site. The second question investigated was whether the known inhibition by acetyl-CoA of binding of NADH, an allosteric inhibitor of E. coli citrate synthase, occurs heterotropically, as an indirect result of acetyl-CoA binding at the active site, or directly, by competition at the allosteric NADH binding site. Using existing restriction sites in the cloned E. coli citrate synthase gene, we prepared a deletion mutant which lacked 24 amino acids near what is predicted to the acetyl-CoA-binding portion of the active site. The mutant protein was inactive, and acetyl-CoA did not bind to the active site but still inhibited NADH binding. Thus acetyl-CoA can interact with both the allosteric and the active sites of this enzyme.     

The role of cysteine 206 in allosteric inhibition of Escherichia coli citrate synthase. Studies by chemical modification, site-directed mutagenesis, and 19F NMR

Escherichia coli citrate synthase is strongly and specifically inhibited by NADH, but this inhibition can be prevented by reacting the enzyme with Ellman's reagent. We have now labeled the single reactive cysteine covalently with monobromobimane and isolated and sequenced the bimane-containing cyanogen bromide peptide and identified the cysteine as Cys-206. Modeling studies suggest that this residue is on the subunit surface, 25-30 A from the active site. Mutation of Cys-206 to serine (C206S), or of Gly-207 to alanine (E207A), weakened NADH binding and inhibition; when these mutations were present together, NADH binding was weaker by 18-fold and inhibition by 250-fold. The mutations also had small effects on substrate binding at the active site. Cys-206 of wild type enzyme and of the mutant E207A was alkylated with 1,1,1-trifluorobromoacetone and the environment of the fluorine nuclei studied by 19F NMR. With wild type enzyme, the NMR spectrum consisted of two peaks of about equal intensity but different line widths, at -8.65 ppm (line width 11.2 +/- 0.5 Hz) and -7.6 ppm (line width 57 +/- 4 Hz). As the labeled wild type citrate synthase was titrated with KCl, the narrow peak converted to the broad one. The same range of KCl concentrations was needed for this conversion as for the allosteric activation of E. coli citrate synthase. The E207A mutant gave the broader NMR peak almost exclusively. We propose that the fluorine label in wild type citrate synthase exists in two conformational states with different mobilities, exchanging slowly on the NMR time scale, and that treatment with KCl, or truncation of the Glu-207 side chain by mutagenesis, stabilizes one of these states. Consistent with this explanation is the finding that Cys-206 reacts more quickly with Ellman's reagent in the presence of KCl, and that this rate is faster yet in the E207A mutant.     

A mutant of Escherichia coli citrate synthase that affects the allosteric equilibrium.

We describe a mutant of Escherichia coli citrate synthase, CS R319L, in which the arginine residue at position 319 of the sequence has been replaced by leucine. In this mutant, saturation by the substrate acetyl-CoA is changed from sigmoid (Hill parameter = 1.75 +/- 0.2) to hyperbolic (Hill parameter = 1.0 +/- 0.1) and dependence on the activator KCl is greatly reduced. Further mutations at this site and at position 343 (which model building predicts is close enough to allow a side-chain interaction with position 319) are also described. In the wild-type enzyme, the model suggests the possibility of a salt-bridge interaction between Arg-319 (located on the P helix in the small domain) and Glu-343 (in the Q helix in the same domain), but mutation of Glu-343 to Ala (CS E343A) produced a much smaller difference in the kinetic properties than the ARg-319 to Leu mutation did. Small changes in kinetic properties were also obtained with an Arg-319----Glu (CS R319E) mutation. In CS R319L, oxaloacetate, the first substrate to bind, induces an ultraviolet difference spectrum which is obtained with wild-type enzyme only in the presence of KCl. To account for these observations we postulate that wild-type E. coli citrate synthase exists in two conformational states, T and R, which are equilibrium; T state binds NADH, the allosteric inhibitor, while R state binds substrates and can be converted to another substrate-binding state, R', by KCl.(ABSTRACT TRUNCATED AT 250 WORDS)     

A comparison of the citrate synthases of Escherichia coli and Acinetobacter anitratum

Citrate synthase has been purified to homogeneity from a strain of the Gram-negative aerobic bacterium Acinetobacter anitratum in a form which retains its sensitivity to the allosteric inhibitor NADH. In subunit size, amino acid composition, and antigenic reactivity the enzyme shows a marked structural resemblance to the citrate synthase of the Gram-negative facultative anaerobe Escherichia coli. Whereas the E. coli enzyme is subject to a strong, hyperbolic inhibition by NADH (Hill's number n = 1.0, Ki = 2 microM), the A. anitratum enzyme shows a weak, sigmoid response (n = 1.6, I0.5 = 140 microM) to this nucleotide. With E. coli, NADH inhibition is competitive with acetyl-CoA, and noncompetitive with oxaloacetate; with A. anitratum, NADH is noncompetitive with both substrates. Acinetobacter anitratum citrate synthase shows hyperbolic saturation with acetyl-CoA (n = 1.8). The finding of Weitzman and Jones (Nature (London) 219, 270 (1968) that NADH inhibition of the enzyme from Acinetobacter spp. is reversible by AMP, while that from E. coli is not, is explained by the much greater affinity of the E. coli enzyme for NADH. Unlike E. coli citrate synthase, the A. anitratum enzyme does not react with the sulfhydryl reagent 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) in the absence of denaturation. With a second sulfhydryl reagent, 4,4'-dithiodipyridine (4,4'-PDS), the A. anitratum enzyme reacts with 1 equiv. of subunit; this modification induces a partial activity loss (attributable to a arise in the Km for acetyl-CoA) and an increase in the sensitivity to NADH. With the E. coli enzyme, 4,4'-PDS causes complete inactivation. Acinetobacter anitratum citrate synthase is much more resistant to urea denaturation than the E. coli enzyme is; the resistance of both enzymes to urea is greatly improved in the presence of 1 M KCl. It is suggested that the amino acid sequences of the subunits of the citrate synthases of these two bacteria are about 90% homologous, and that the 10% differences are in key residues, perhaps largely in the subunit contact regions, which account for the differences in allosteric properties.     

Structure of a NADH-insensitive hexameric citrate synthase that resists acid inactivation

Acetobacter aceti converts ethanol to acetic acid, and strains highly resistant to both are used to make vinegar. A. aceti survives acetic acid exposure by tolerating cytoplasmic acidification, which implies an unusual adaptation of cytoplasmic components to acidic conditions. A. aceti citrate synthase (AaCS), a hexameric type II citrate synthase, is required for acetic acid resistance and, therefore, would be expected to function at low pH. Recombinant AaCS has intrinsic acid stability that may be a consequence of strong selective pressure to function at low pH, and unexpectedly high thermal stability for a protein that has evolved to function at approximately 30 degrees C. The crystal structure of AaCS, complexed with oxaloacetate (OAA) and the inhibitor carboxymethyldethia-coenzyme A (CMX), was determined to 1.85 A resolution using protein purified by a tandem affinity purification procedure. This is the first crystal structure of a "closed" type II CS, and its active site residues interact with OAA and CMX in the same manner observed in the corresponding type I chicken CS.OAA.CMX complex. While AaCS is not regulated by NADH, it retains many of the residues used by Escherichia coli CS (EcCS) for NADH binding. The surface of AaCS is abundantly decorated with basic side chains and has many fewer uncompensated acidic charges than EcCS; this constellation of charged residues is stable in varied pH environments and may be advantageous in the A. aceti cytoplasm.     

Comparative analysis of folding and substrate binding sites between regulated hexameric type II citrate synthases and unregulated dimeric type I enzymes.

We describe the first structure determination of a type II citrate synthase, an enzyme uniquely found in Gram-negative bacteria. Such enzymes are hexameric and are strongly and specifically inhibited by NADH through an allosteric mechanism. This is in contrast to the widespread dimeric type I citrate synthases found in other organisms, which do not show allosteric properties. Our structure of the hexameric type II citrate synthase from Escherichia coli is composed of three identical dimer units arranged about a central 3-fold axis. The interactions that lead to hexamer formation are concentrated in a relatively small region composed of helix F, FG and IJ helical turns, and a seven-residue loop between helices J and K. This latter loop is present only in type II citrate synthase sequences. Running through the middle of the hexamer complex, and along the 3-fold axis relating dimer units, is a remarkable pore lined with 18 cationic residues and an associated hydrogen-bonded network. Also unexpected was the observation of a novel N-terminal domain, formed by the collective interactions of the first 52 residues from the two subunits of each dimer. The domain formed is rich in beta-sheet structure and has no counterpart in previous structural studies of type I citrate synthases. This domain is located well away from the dimer-dimer contacts that form the hexamer, and it is not involved in hexamer formation. Another surprising observation from the structure of type II E. coli citrate synthase is the unusual polypeptide chain folding found at the putative acetylcoenzyme A binding site. Key parts of this region, including His264 and a portion of polypeptide chain known from type I structures to form an adenine binding loop (residues 299-303), are shifted by as much as 10 A from where they must be for substrate binding and catalysis to occur. Furthermore, the adjacent polypeptide chain composed of residues 267-297 is extremely mobile in our structure. Thus, acetylcoenzyme A binding to type II E. coli citrate synthase would require substantial structural shifts and a concerted refolding of the polypeptide chain to form an appropriate binding subsite. We propose that this essential rearrangement of the acetylcoenzyme A binding part of the active site is also a major feature of allostery in type II citrate synthases. Overall, this study suggests that the evolutionary development of hexameric association, the elaboration of a novel N-terminal domain, introduction of a NADH binding site, and the need to refold a key substrate binding site are all elements that have been developed to allow for the allosteric control of catalysis in the type II citrate synthases.     

Expression and base sequence of the citrate synthase gene of Acinetobacter anitratum

The sequence of 1895 base pairs of Acinetobacter anitratum genomic DNA, containing the structural gene for the allosteric citrate synthase of that Gram-negative bacterium, is presented. The sequence contains an open reading frame of 424 codons, the 5' end of which is the same as the N-terminal sequence of A. anitratum citrate synthase, less the initiator methionine. The inferred amino acid sequence of the enzyme is about 70% identical with that of citrate synthase from Escherichia coli, which like the A. anitratum enzyme is sensitive to allosteric inhibition by NADH. There is also a more distant homology with the nonallosteric citrate synthases of pig heart and yeast. The gene contains sequences that strongly resemble those found in E. coli promoters, an E. coli type of ribosomal binding site, and a hyphenated dyad sequence at the 3' end of the gene which resembles the rho-independent terminators found in some E. coli genes. The plasmid clone containing the A. anitratum citrate synthase gene pLJD1, originally isolated because it hybridized with the cloned E. coli citrate synthase gene under conditions of reduced stringency, produces large amounts of A. anitratum citrate synthase in an E. coli host which lacks citrate synthase. This work completes proof of the hypothesis that the three major kinds of citrate synthases are formed of similar subunits, although their functional properties are different.     

Biosynthesis of bacterial glycogen. Incorporation of pyridoxal phosphate into the allosteric activator site and an ADP-glucose-protected pyridoxal phosphate binding site of Escherichia coli B ADP-glucose synthase.

[3H]Pyridoxal-P can be covalently incorporated into Escherichia coli B mutant strain AC70R1 ADP-glucose synthase by reduction with NaBH4. Two distinct lysine residues can be modified by the allosteric activator pyridoxal-P. Incorporation of [3H]pyridoxal-P in the presence of substrate ADP-glucose + MgCl2 prevents pyridoxylation of an ADP-glucose-protected site and allows modification of the allosteric activator site. Incorporation of [3H]pyridoxal-P in the presence of allosteric effectors fructose-P2, 5'-AMP, or hexanediol-1,6-P2, protects against pyridoxylation of the allosteric activator site, and allows modification of the ADP-glucose-protected site. Incorporation of pyridoxal-P into the allosteric activator site results in modified enzyme of high activity form, even in the absence of fructose-P2. This modified enzyme, when assayed in the absence of fructose-P2, exhibits activation kinetics similar to nonpyridoxylated enzyme assayed in the presence of fructose-P2 and is still inhibited by 5'-AMP. These data suggest that the allosteric activator site of pyridoxylation is the fructose-P2 binding site, and is distinct from the inhibitor 5'-AMP binding site. Incorporation of pyridoxal-P into the ADP-glucose-protected site results in a decrease in enzyme activity. This pyridoxylated lysine could be involved with the binding of thesubstrates ADP-glucose, alpha-glucose-1-P, or PPi, or participate in the catalytic mechanism of the enzyme.     

Probing the roles of key residues in the unique regulatory NADH binding site of type II citrate synthase of Escherichia coli

The citrate synthase of Escherichia coli is an example of a Type II citrate synthase, a hexamer that is subject to allosteric inhibition by NADH. In previous crystallographic work, we defined the NADH binding sites, identifying nine amino acids whose side chains were proposed to make hydrogen bonds with the NADH molecule. Here, we describe the functional properties of nine sequence variants, in which these have been replaced by nonbonding residues. All of the variants show some changes in NADH binding and inhibition and small but significant changes in kinetic parameters for catalysis. In three cases, Y145A, R163L, and K167A, NADH inhibition has become extremely weak. We have used nanospray/time-of-flight mass spectrometry, under non-denaturing conditions, to show that two of these, R163L and K167A, do not form hexamers in response to NADH binding, unlike the wild type enzyme. One variant, R109L, shows tighter NADH binding. We have crystallized this variant and determined its structure, with and without bound NADH. Unexpectedly, the greatest structural changes in the R109L variant are in two regions outside the NADH binding site, both of which, in wild type citrate synthase, have unusually high mobilities as measured by crystallographic thermal factors. In the R109L variant, both regions (residues 260 -311 and 316-342) are much less mobile and have rearranged significantly. We argue that these two regions are elements in the path of communication between the NADH binding sites and the active sites and are centrally involved in the regulatory conformational change in E. coli citrate synthase.     

Evidence of isosteric and allosteric nucleotide inhibition of citrate synthease from multiple-inhibition studies.

Citrate synthases from diverse organisms are inhibited by ATP and NADH. Evidence is presented, from multiple-inhibition studies on various citrate synthases, that ATP acts in all cases as an isosteric inhibitor at the acetyl-CoA site. On the other hand, NADH also acts isosterically with eukaryotic and Gram-positive bacterial citrate synthases, but behaves as an allosteric inhibitor specifically in the case of the Gram-negative bacterial enzyme. After desensitization to this allosteric inhibition, only the isosteric nucleotide inhibition, as found in other citrate syntheases, is observed.     

Dissection of the effector-binding site and complementation studies of Escherichia coli phosphofructokinase using site-directed mutagenesis

A systematic study by site-directed mutagenesis has been conducted on the effector site of phosphofructokinase from Escherichia coli to delineate the role of side chains in binding the allosteric activator, GDP, and inhibitor, PEP, and to search for key residues in the allosteric transtion. Target residues were identified from the crystal structure of the enzyme-nucleoside diphosphate complex. It is found that both activator and inhibitor bind to the same set of amino acid side chains. Deletion of positively charged groups (Arg21, Arg25, Arg54, Arg154, and Lys213 mutated to alanine) weakens binding of both effectors by 2-3 kcal/mol, consistent with the disruption of charged hydrogen bonds. Residue Glu187, which is known from the crystal structure to bind the coordinated Mg2+ ion of GDP, is found to have a unique behavior on mutation and appears to be crucial in triggering the allosteric transition. All other residues mutated simply weaken binding of both PEP and GDP in a parallel manner. However, mutation of Glu----Ala187 reverses the roles of GDP and PEP, causing GDP to become an allosteric inhibitor and PEP an activator. Mutation of Glu----Gln187 has only a small effect on the binding of PEP, and both PEP and GDP are inhibitors. Studies are described in which mutations in different subunits of a tetrameric complex complement each other. The effector site is composed of residues from two subunits. In particular, Arg21 and Lys213 in each site are from different subunits. Mutations of either one of these residues abolishes activation by GDP of the homotetramer.(ABSTRACT TRUNCATED AT 250 WORDS)     

Comparison of AMP and NADH binding to glycogen phosphorylase b

The binding sites for the allosteric activator, AMP, to glycogen phosphorylase b are described in detail utilizing the more precise knowledge of the native structure obtained from crystallographic restrained least-squares refinement than has hitherto been available. Localized conformational changes are seen at the allosteric effector site that include shifts of between 1 and 2 A for residues Tyr75 and Arg309 and very small shifts for the region of residues 42 to 44 from the symmetry-related subunit. Kinetic studies demonstrate that NADH inhibits the AMP activation of glycogen phosphorylase b. Crystallographic binding studies at 3.5 A resolution show that NADH binds to the same sites on the enzyme as AMP, i.e. the allosteric effector site N, which is close to the subunit-subunit interface, and the nucleoside inhibitor site I, which is some 12 A from the catalytic site. The conformations of NADH at the two sites are different but both conformations are "folded" so that the nicotinamide ring is close (approx. 6 A) to the adenine ring. These conformations are compared with those suggested from solution studies and with the extended conformations observed in the single crystal structure of NAD+ and for NAD bound to dehydrogenases. Possible mechanisms for NADH inhibition of phosphorylase activation are discussed.     

Interaction of glycogen synthase from rabbit skeletal muscles with 1,5-gluconolactone

1.5-Gluconolactone was shown to inhibit in a competitive manner the activity of both I- and D-forms of rabbit skeletal muscle glycogen synthase. Unlike other known inhibitors (UDP and adenyl nucleotides) the affinity of the enzyme D-form for 1.5-gluconolactone is lower than that of the I-form. The joint inhibition of glycogen synthase by UDP and 1.5-gluconolactone is characterized by positive cooperativity. It was supposed that the binding of the nucleotide part of the substrate molecule is preceded by the UDPglucose glucosyl residue interaction with the enzyme and induces a closer resemblance to the transient state. The effect of the allosteric inhibitor, ADP, on the enzyme activity is conditioned by its effect on the conformational state of UDP-glucose glucosyl residue binding site. Phosphorylation of glycogen synthase results in conformational changes in the same active site region, although the pyrimidine base binding site also seems to be involved in this process.     

Biochemical and Kinetic Characterization of Xylulose 5-Phosphate/Fructose 6-Phosphate Phosphoketolase 2 (Xfp2) from Cryptococcus neoformans

Xylulose 5-phosphate/fructose 6-phosphate phosphoketolase (Xfp), previously thought to be present only in bacteria but recently found in fungi, catalyzes the formation of acetyl phosphate from xylulose 5-phosphate or fructose 6-phosphate. Here, we describe the first biochemical and kinetic characterization of a eukaryotic Xfp, from the opportunistic fungal pathogen Cryptococcus neoformans, which has two XFP genes (designated XFP1 and XFP2). Our kinetic characterization of C. neoformans Xfp2 indicated the existence of both substrate cooperativity for all three substrates and allosteric regulation through the binding of effector molecules at sites separate from the active site. Prior to this study, Xfp enzymes from two bacterial genera had been characterized and were determined to follow Michaelis-Menten kinetics. C. neoformans Xfp2 is inhibited by ATP, phosphoenolpyruvate (PEP), and oxaloacetic acid (OAA) and activated by AMP. ATP is the strongest inhibitor, with a half-maximal inhibitory concentration (IC₅₀) of 0.6 mM. PEP and OAA were found to share the same or have overlapping allosteric binding sites, while ATP binds at a separate site. AMP acts as a very potent activator; as little as 20 μM AMP is capable of increasing Xfp2 activity by 24.8% ± 1.0% (mean ± standard error of the mean), while 50 μM prevented inhibition caused by 0.6 mM ATP. AMP and PEP/OAA operated independently, with AMP activating Xfp2 and PEP/OAA inhibiting the activated enzyme. This study provides valuable insight into the metabolic role of Xfp within fungi, specifically the fungal pathogen Cryptococcus neoformans, and suggests that at least some Xfps display substrate cooperative binding and allosteric regulation.     

Overall kinetic mechanism of saccharopine dehydrogenase from Saccharomyces cerevisiae

Kinetic data have been measured for the histidine-tagged saccharopine dehydrogenase from Saccharomyces cerevisiae, suggesting the ordered addition of nicotinamide adenine dinucleotide (NAD) followed by saccharopine in the physiologic reaction direction. In the opposite direction, the reduced nicotinamide adenine dinucleotide (NADH) adds to the enzyme first, while there is no preference for the order of binding of alpha-ketoglutarate (alpha-Kg) and lysine. In the direction of saccharopine formation, data also suggest that, at high concentrations, lysine inhibits the reaction by binding to free enzyme. In addition, uncompetitive substrate inhibition by alpha-Kg and double inhibition by NAD and alpha-Kg suggest the existence of an abortive E:NAD:alpha-Kg complex. Product inhibition by saccharopine is uncompetitive versus NADH, suggesting a practical irreversibility of the reaction at pH 7.0 in agreement with the overall K(eq). Saccharopine is noncompetitive versus lysine or alpha-Kg, suggesting the existence of both E:NADH:saccharopine and E:NAD:saccharopine complexes. NAD is competitive versus NADH, and noncompetitive versus lysine and alpha-Kg, indicating the combination of the dinucleotides with free enzyme. Dead-end inhibition studies are also consistent with the random addition of alpha-Kg and lysine. Leucine and oxalylglycine serve as lysine and alpha-Kg dead-end analogues, respectively, and are uncompetitive against NADH and noncompetitive against alpha-Kg and lysine, respectively. Oxaloacetate (OAA), pyruvate, and glutarate behave as dead-end analogues of lysine, which suggests that the lysine-binding site has a higher affinity for keto acid analogues than does the alpha-Kg site or that dicarboxylic acids have more than one binding mode on the enzyme. In addition, OAA and glutarate also bind to free enzyme as does lysine at high concentrations. Glutarate gives S-parabolic noncompetitive inhibition versus NADH, indicating the formation of a E:(glutarate)2 complex as a result of occupying both the lysine- and alpha-Kg-binding sites. Pyruvate, a slow alternative keto acid substrate, exhibits competitive inhibition versus both lysine and alpha-Kg, suggesting the combination to the E:NADH:alpha-Kg and E:NADH:lysine enzyme forms. The equilibrium constant for the reaction has been measured at pH 7.0 as 3.9 x 10(-7) M by monitoring the change in NADH upon the addition of the enzyme. The Haldane relationship is in very good agreement with the directly measured value.     

Chimeric allosteric citrate synthases: construction and properties of citrate synthases containing domains from two different enzymes.

The citrate synthases of the gram-negative bacteria, Escherichia coli and Acinetobacter anitratum, are allosterically inhibited by NADH. The kinetic properties, however, suggest that the equilibrium between active (R) and inactive (T) conformational states is shifted toward the T state in the E. coli enzyme. We have now manipulated the cloned genes for the two bacterial enzymes to produce two chimeric proteins, in which one folding domain of each subunit is derived from each enzyme. One chimera (the large domain from A. anitratum and the small domain from the E. coli enzyme) is designated CS ACI::eco; the other is called CS ECO::aci. Both chimeras are roughly as active as the wild type parents, but their Km values for both substrates are lower than those for the E. coli enzyme, and NADH inhibition is markedly sigmoid, while that for E. coli citrate synthases is hyperbolic. Curve-fitting to the allosteric equation suggests that these differences are the result of the destabilization of the T state in the chimeras. The ACI::eco chimera exists almost entirely as a hexamer, like the A. anitratum enzyme, while the ECO::aci chimera, like the E. coli synthase, forms three major bands on nondenaturing polyacrylamide gels, two of them hexamers of different net charge, and one a dimer. These findings indicate that subunit interactions leading to hexamer formation in allosteric citrate synthases of gram-negative bacteria involve mainly the large domains. The chimeras are also used to show that the NADH binding site of E. coli citrate synthase is located entirely in the large domain. Sensitivity of the chimeras to denaturation by urea, to which the A. anitratum enzyme is much more resistant than the E. coli enzyme, is determined by the large domains. Sensitivity to inactivation by subtilisin is intermediate between those shown by the E. coli (very sensitive) and A. anitratum (quite resistant) synthases. This result suggests that digestibility by subtilisin is determined by conformational factors as well as the amino acid sequences of the target regions.     

Nucleotide binding to glycogen phosphorylase b in the crystal.

The binding of the allosteric activator, AMP, and the inhibitor, ATP, to glycogen phosphorylase b has been studied in the crystal at 3 Å resolution. The nucleotides bind to two sites on the enzyme which are identified as site N, the allosteric effector site which is close to the subunit-subunit interface, and site I, a nucleoside inhibitor site which blocks the entrance to the active site crevasse. AMP when bound at the allosteric effector site makes several defined interactions with the enzyme in agreement with the results of solution studies. The contacts involve the N-10 position of the base, the 2′ hydroxyl of the ribose and the phosphate. IMP, analysed at 4 Å resolution, appears to bind in an identical conformation to AMP. At 3 Å resolution no well defined conformational changes are observed on binding AMP, although there are indications of a disturbance of the crystal lattice. It is concluded that the forces which stabilise the crystal lattice prevent the allosteric response of the enzyme in the crystal.     

Three-dimensional structure of the R115E mutant of T4-bacteriophage 2'-deoxycytidylate deaminase

2'-Deoxycytidylate deaminase (dCD) converts deoxycytidine 5'-monophosphate (dCMP) to deoxyuridine 5'-monophosphate and is a major supplier of the substrate for thymidylate synthase, an important enzyme in DNA synthesis and a major target for cancer chemotherapy. Wild-type dCD is allosterically regulated by the end products of its metabolic pathway, deoxycytidine 5'-triphosphate and deoxythymidine 5'-triphosphate, which act as an activator and an inhibitor, respectively. The first crystal structure of a dCD, in the form of the R115E mutant of the T4-bacteriophage enzyme complexed with the active site inhibitor pyrimidin-2-one deoxyribotide, has been determined at 2.2 A resolution. This mutant of dCD is active, even in the absence of the allosteric regulators. The molecular topology of dCD is related to that of cytidine deaminase (CDA) but with modifications for formation of the binding site for the phosphate group of dCMP. The enzyme has a zinc ion-based mechanism that is similar to that of CDA. A second zinc ion that is present in bacteriophage dCD, but absent in mammalian dCD and CDA, is important for the structural integrity of the enzyme and for the binding of the phosphate group of the substrate or inhibitor. Although the R115E mutant of dCD is a dimer in solution, it crystallizes as a hexamer, mimicking the natural state of the wild-type enzyme. Residues 112 and 115, which are known to be important for the binding of the allosteric regulators, are found in a pocket that is at the intersubunit interfaces in the hexamer but distant from the substrate-binding site. The substrate-binding site is composed of residues from a single protein molecule and is sequestered in a deep groove. This groove is located at the outer surface of the hexamer but ends at the subunit interface that also includes residue 115. It is proposed that the absence of subunit interactions at this interface in the dimeric R115E mutant renders the substrate-binding site accessible. In contrast, for the wild-type enzyme, binding of dCTP induces an allosteric effect that affects the subunit interactions and results in an increase in the accessibility of the binding site.