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H W Duckworth

Publications and source records attributed to H W Duckworth.

15 recordsLinked to original sources
PubMed1992

Proton uptake accompanies formation of the ternary complex of citrate synthase, oxaloacetate, and the transition-state analog inhibitor, carboxymethyl-CoA. Evidence that a neutral enol is the activated form of acetyl-CoA in the citrate synthase reaction.

Citrate synthase complexes with the transition-state analog inhibitor, carboxymethyl-CoA (CM-CoA), are believed to mimic those with the activated form of acetyl-CoA. The X-ray structure [Karpusas, M., Branchaud, B., & Remington, S.J. (1990) Biochemistry 29, 2213] of the ternary complex of the enzyme, oxaloacetate, and CMCoA has been used as the basis for a proposal that a neutral enol of acetyl-CoA is that activated form. Since the inhibitor carboxyl has a pKa of 3.90, analogy with an enolic acetyl-CoA intermediate leads to the prediction that a proton should be taken up from solution upon formation of the analog complex so that the transition-state analog carboxyl is protonated when bound. We have obtained evidence in solution for this proposal by comparing the isoelectric points and the pH dependence of the dissociation constants of the ternary complexes of the pig heart enzyme with the neutral ground-state analog inhibitor, acetonyl-CoA (KCoA), and the anionic transition-state analog inhibitor (CMCoA) and by studying the NMR spectra of the transition-state analog complexes of allosteric (Escherichia coli) and nonallosteric (pig heart) enzymes. The pH dependence of the dissociation constant of the ground-state analog indicates no proton uptake, while that for the transition-state analog indicates that 0.55 +/- 0.04 proton is taken up when the analog binds to the citrate synthase-oxaloacetate binary complex. The overall charges of ternary complexes of the pig heart enzyme with the transition-state and ground-state analog inhibitors are the same, as monitored by their isoelectric points.(ABSTRACT TRUNCATED AT 250 WORDS)

Acetyl Coenzyme A↗
PubMed1992

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.

Acinetobacter↗
PubMed1991

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.

Alkylation↗
PubMed1991

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)

Allosteric Regulation↗
PubMed1991

Sequences of the variable regions of three monoclonal antibodies specific for histidine-containing protein of the bacterial phosphoenolpyruvate:sugar phosphotransferase system.

The variable regions of three monoclonal antibodies, Jel 42, Jel 44, and Jel 324, specific for the histidine-containing protein of the bacterial phosphoenolpyruvate:sugar phosphotransferase system have been sequenced from their respective mRNAs. The Vh gene families were deduced from the percent homology to the concensus gene sequences and the J gene and D gene usage was also analysed.

Amino Acid Sequence↗
PubMed1989

Cloning, sequencing, and expression of the gene for NADH-sensitive citrate synthase of Pseudomonas aeruginosa.

The structural gene for the allosteric citrate synthase of Pseudomonas aeruginosa has been cloned from a genomic library by using the Escherichia coli citrate synthase gene as a hybridization probe under conditions of reduced stringency. Subcloning of portions of the original 10-kilobase-pair (kbp) clone led to isolation of the structural gene, with its promoter, within a 2,083-bp length of DNA flanked by sites for KpnI and BamHI. The nucleotide sequence of this fragment is presented; the inferred amino acid sequence was 70 and 76% identical, respectively, with the citrate synthase sequences from E. coli and Acinetobacter anitratum, two other gram-negative bacteria. DEAE-cellulose chromatography of P. aeruginosa citrate synthase from an E. coli host harboring the cloned P. aeruginosa gene gave three peaks of activity. All three enzyme peaks had subunit molecular weights of 48,000; the proteins were identical by immunological criteria and very similar in kinetics of substrate saturation and NADH inhibition. Because the cloned gene contained only one open reading frame large enough to encode a polypeptide of such a size, the three peaks must represent different forms of the same protein. A portion of the cloned P. aeruginosa gene was used as a hybridization probe under stringent conditions to identify highly homologous sequences in genomic DNA of a second strain classified as P. aeruginosa and isolates of P. putida, P. stutzeri, and P. alcaligenes. When crude extracts of each of these four isolates were mixed with antiserum raised against purified P. aeruginosa citrate synthase, however, only the P. alcaligenes extract cross-reacted.

Amino Acid Sequence↗
PubMed1988

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.

Binding Sites↗
PubMed1987

Structural basis for regulation in gram-negative bacterial citrate synthases.

The citrate synthases of Gram-negative bacteria, unlike those of eukaryotes, are inhibited allosterically by NADH, but the two kinds of citrate synthase are about 30% homologous in amino acid sequence--the two Gram-negative citrate synthase sequences so far determined, from Escherichia coli and Acinetobacter anitratum, are about 70% identical. A model for the NADH-sensitive E. coli citrate synthase has been constructed using sequence homology and the known structure of the pig heart enzyme. The most reactive cysteine in the E. coli enzyme, which probably marks the NADH binding site, has now been identified as Cys-206. The model places this residue far from the active site. An E. coli citrate synthase mutant, from which a stretch of 24 amino acids has been deleted near the active site, still binds NADH normally. Two active site missense mutants of this enzyme, generated by oligonucleotide-directed mutagenesis, have lower affinities for one substrate, oxaloacetate, but also are much less sensitive to 2-oxoglutarate, an oxaloacetate analogue hitherto believed to be an allosteric inhibitor. These results confirm that NADH binds to a truly allosteric site in E. coli citrate synthase, the features of which are still to be defined; while 2-oxoglutarate is really an active-site directed inhibitor, although it may still play a regulatory role in vivo.

Binding Sites↗
PubMed1986

Molecular cloning of the structural gene for Acinetobacter citrate synthase.

The structural gene for citrate synthase of Acinetobacter anitratum has been cloned in Escherichia coli in a form which expresses the enzyme. A library of EcoRI fragments of Acinetobacter genomic DNA was prepared in the vector lambda gt10, and clones were screened by hybridization with an E. coli citrate synthase clone under conditions of reduced stringency. A 6.5 kbp clone was obtained which was subcloned into pBR322, and shown to direct the formation of Acinetobacter citrate synthase in E. coli hosts. The promoter was located within a BglII fragment, and from this information the orientation of the gene was deduced.

Acinetobacter↗
PubMed1979

The interactions of adenylates with allosteric citrate synthase.

Evidence is presented that a number of derivatives of adenylic acid may bind to the allosteric NADH binding site of Escherichia coli citrate synthase. This evidence includes the facts that all the adenylates inhibit NADH binding in a competitive manner and that those which have been tested protect an enzyme sulfhydryl group from reaction with 5,5'-dithiobis-(2-nitrobenzoic acid) in the same way that NADH does. However, whereas NADH is a potent inhibitor of citrate synthase, most of the adenylates are activators. The best activator, ADP-ribose, increases the affinity of the enzyme for the substrate, acetyl-CoA, and saturates the enzyme in a sigmoid manner. A fluorescence technique, involving the displacement of 8-anilino-1-naphthalenesulfonate from its complex with citrate synthase, is used to obtain saturation curves for several nucleotides under nonassay conditions. It is found that acetyl-coenzyme A, coenzyme A, and ADP-ribose all bind to the enzyme cooperatively, and that the binding of each becomes tighter in the presence of KCl, the activator, and oxaloacetic acid (OAA), the second substrate. Another inhibitor, alpha-ketoglutarate, can complete with OAA in the absence of KCl but not in its presence. The nature of the allosteric site of citrate synthase, and the modes of action of several activators and inhibitors, are discussed in the light of this evidence.

Adenine Nucleotides↗
PubMed1979

The reactions of Escherichia coli citrate synthase with the sulfhydryl reagents 5,5'-dithiobis-(2-nitrobenzoic acid) and 4,4'-dithiodipyridine.

Citrate synthase of Escherichia coli reacts rapidly with 1 equivalent of Ellman's reagent, 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB), per subunit, losing completely its sensitivity to the allosteric inhibitor, NADH. When the enzyme is treated instead with 4,4'-dithiodipyridine (4,4'-PDS), all activity is lost. Certain evidence in this paper is consistent with the belief that the sulfhydryl group modified by DTNB, and that whose modification by 4,4'-PDS inactivates the enzyme, are the same. (i) Both reagents abolish NADH fluorescence enhancement by the enzyme. (ii) Saturating levels of NADH and some other adenylic acid derivatives inhibit the reactions with both reagents. (iii) When the enzyme is modified with one equivalent of DTNB or 4,4'-PDS, subsequent reactivity toward the other reagent is greatly decreased. (iv) Following modifications, the DTNB and 4,4'-PDS derivatives spontaneously lose thionitrobenzoate (TNB) or pyridine-4-thione (PT), respectively, in reactions which are thought to involve displacement of TNB or PT by a second enzyme sulfhydryl group, so that an enzyme disulfide is introduced. The introduction of the disulfide bond, if this is what occurs, does not lead to cross-linking of citrate synthase polypeptide chains, as judged by sodium dodecyl sulfate polyacrylamide gel electrophoresis under nonreducing conditions. Certain evidence has also been found, however, that the sites of modification by DTNB and 4,4'-PDS are not the same. (i) DTNB modification desensitizes to NADH but does not inactivate, while 4,4'-PDS inactivates at least 99.9%. (ii) The presumed disulfide from elimination of TNB is also active, while that from PT modification is no more active than the original 4,4'-PDS modified product. (iii) Prior modification of the enzyme with DTNB affords no protection against later inactivation by 4,4'-PDS. The studies therefore indicate a close relationship between the DTNB desensitization and 4,4'-PDS inactivation, but they are unable to identify it exactly. Other properties of the DTNB reaction are also described, and a hypothesis is offered to explain quantitatively the finding that desensitization lags behind modification during the modification of citrate synthase by DTNB.

Binding Sites↗
PubMed1976

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.

Binding Sites↗
PubMed1975

The quaternary structure of citrate synthase from Escherichia coli K12.

A combination of equilibrium ultracentrifugation and polyacrylamide gel electrophoresis techniques has been used to establish the quaternary structure of citrate synthase from acetate-grown Escherichia coli K12 3000. In polyacrylamide gels containing 0.1% sodium dodecyl sulfate (SDS), the pure enzyme showed one major band whose mobility was consistent with a molecular weight of 46,000 plus or minus 2000 g/mol, and a little material of 87,000 plus or minus 5000 g/mol. When first cross-linked with dimethyl suberimidate and then submitted to electrophoresis in SDS, citrate synthase showed six bands, in widely different amounts, whose apparent molecular weights were almost integral multiples of 47,000 g/mol. The dimer was the major product of the cross-linking procedure. In 6 M guanidine HCl at pH 7.0, citrate synthase behaved as a single component in high-speed sedimentation equilibrium experiments, with a weight average molecular weight of 43,400 plus or minus 300 g/mol. The molecular weight of native citrate synthase was investigated by high-speed sedimentation equilibrium ultracentrifugation under different conditions of pH and KCl concentration. In 0.02 M Tris-Cl at pH 7.0 and 7.8, the enzyme was a mixture of oligomers, with species ranging from monomer (47,000 g/mol) to greater than decamer being present. At pH 9.0, only dimer was seen (94,000 g/mol). Large aggregates were present at pH 10.0. The addition of small amounts of KCl, a potent activator of the enzyme, simplified the mixture of oligomers considerably at pH 7.8. A detailed analysis of the data with 0.05 M KCl indicated that dimer and hexamer were the only species present, with marked nonideality. Increasing the KCl concentration to 0.10 M converted all the enzyme to hexamer. The amino acid composition of E. coli citrate synthase was presented. Taken together with peptide mapping experiments of others (J. A. Wright and B. D. Sanwal (1971), J. Biol. Chem. 246 1689), it indicates that the subunits have all the same or very similar amino acid sequences. The dansylation method revealed only methionine at the N-termini of the citrate synthase polypeptide chains. Citrate synthase from E. coli thus resembles the enzyme from eukaryotes in that it consists of subunits weighing just under 50,000 g/mol, although these subunits are more highly aggregated in the bacterial enzyme under most conditions. This conclusion is in disagreement with that of Wright and Sanwal (1971, see above), who reported a subunit size of 62,000 g/mol.

Amino Acids↗
PubMedDate not supplied

Mutation of amino acids thought to polarize the oxaloacetate carbonyl in citrate synthase severely reduces but does not abolish activity of the enzyme.

Oligonucleotide-directed mutagenesis has been used to alter two active site residues of Escherichia coli citrate synthase, histidine-305 and arginine-314. Both residues are thought to be involved in the polarization of the carbonyl group of oxaloacetate and thus facilitate attack at the carbonyl carbon by acetyl-CoA. In one mutant, designated CS305H----A, His-305 was mutated to alanine and in the other, designated CS314R----L, Arg-314 was changed to leucine. Both mutants have greatly reduced turnover numbers, less than 0.1% of the wild-type value. The dissociation constant for formation of the binary enzyme-oxaloacetate complex, Ki, OAA, is at least 950 microM for CS305H----A, and about 500 microM for CS314R----L, 28 and 15 times the wild-type value, respectively. The Michaelis constants for the two substrates, KOAA and KAcCoA, which measure the affinity of the enzyme for the catalytically significant ternary complex, are less radically altered: values of KAcCoA are actually 3.5-fold and 4.6-fold lower for CS305H----A and CS314R----L, respectively. These kinetic effects are taken to mean that both His-305 and Arg-314 are important for the successful formation of an efficient transition state, very likely by polarizing the carbonyl group of oxaloacetate as has been suggested, and that the residual kinetic activity, in both mutants, occurs by a mechanism which benefits from only part of this polarization. Allosteric properties of the mutant enzymes, as measured by NADH inhibition and binding, and KCl activation, are normal.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acids↗