Coenzyme A dithioesters: synthesis, characterization and reaction with citrate synthase and acetyl-CoA:choline O-acetyltransferase

Acyl dithioesters of CoA have been synthesized by transesterification. The alpha-hydrogens have a spectrally determined pKa of 12.5 +/- 0.14. The hydroxide catalyzed enolization rate is estimated to be 600 M-1.s-1. The absorbance of the dithioester, lambda max = 306 nm, can be used to monitor both the condensation and transesterification reactions that use CoA-Ac as a substrate. For citrate synthase at pH 7.4 Vmax = (4.0 +/- 0.4).10(-4) s-1 and Km = 53 +/- 7.5 microM, which are 2.10(-6) and 3.3-times the Vmax and Km values observed for CoAS-Ac, while for Ac-CoA: choline O-acetyltransferase (EC 2.3.1.6) at pH 7.0 Vmax = (1.1 +/- 0.2).10(-2) mumol.s-1.(mg protein)-1 and Km = 83 +/- 33 microM, which are 0.077 and 10-times the values observed with CoAS-Ac, respectively. The CoA dithioesters are stable at low pH, but hydrolyze with a second-order rate constant of 8.2.10(-2) M-1.s-1 at pH 11.4. The spectral properties of these dithioesters should allow these analogs to be used as probes of the structure of enzyme bound intermediates.     

The reaction of acetyldithio-CoA, a readily enolized analog of acetyl-CoA with thiolase from Zoogloea ramigera

Acetyldithio-CoA has been shown to be a competent nucleophilic substrate but not an electrophilic substrate for the Claisen condensation catalyzed by thiolase, which normally dimerizes acetyl (Ac)-CoA to acetoacetyl-CoA. Acting as the nucleophile, the kcat/Km for dithioacetyl-CoA is comparable to that of Ac-CoA, the normal substrate. With acetoacetyl-pantetheine acetylating the thiolase to provide the electrophile, the kcat and kcat/Km for the Claisen condensation are 2.1 s-1 and 8.3 X 10(4) M-1 s-1, respectively. The product of the reaction is 3-ketobutyryldithio-CoA. The 3-ketobutyryldithio-CoA has a spectrally determined pKa of 6.55 and the enolate has a lambda max of 357 nm, epsilon 357 = 21,000 cm-1 M-1. Product analysis indicates that acetyldithio-CoA does not serve as the electrophilic partner in the enzymic condensation. This failure is attributed to the inability demonstrated in this study of acetyldithio-CoA to thioacetylate the active site Cys89 of the Zoogloea ramigera thiolase. 1H NMR studies in D2O indicate that thiolase catalyzes the exchange of the alpha-hydrogens, without Cys89 being acetylated, with a rate of 0.63 +/- 0.25 s-1. In the presence of a large excess of acetoacetyl-pantetheine, present to acetylate Cys89 and prevent the thiolytic back reaction, solvent exchange of the alpha-hydrogens can still be detected by observing the isotope-shifted 13C NMR spectrum of [2-13C]acetyldithio-CoA. The exchange of the acetyldithio-CoA alpha-hydrogens with solvent promoted by the acetylated enzyme, must proceed at a rate comparable to that of the condensation reaction.     

Investigating the role of the geminal dimethyl groups of coenzyme A: synthesis and studies of a didemethyl analogue

An analogue 2 of coenzyme A (CoA) has been prepared in which the geminal methyl groups are replaced with hydrogens. An NMR titration study was conducted and shifts in frequency of protons in the pantetheine portion of the molecule upon titration of the adenine base were observed as has been previously reported with CoA. These studies indicate that the geminal dimethyl groups are not essential for adoption of a partially folded conformation in solution. Based on 1H-1H coupling constants, the distribution of conformations about the carbon-carbon bonds in the region of the methyl deletion were estimated. The results suggest that the conformer distribution is similar to that of CoA, but with small increases in population of the anti conformers. A simple model compound containing the didemethyl pantoamide moiety was prepared and subjected to similar conformational analysis. The coupling constants and predicted conformer distribution were almost identical to that of the CoA analogue, indicating that the conformer distribution is controlled by local interactions and not influenced by interactions between distant parts of the CoA molecule. The acetyl derivative of 2 was a fairly good substrate for the acetyl-CoA utilizing enzymes carnitine acetyltransferase, chloramphenicol acetyltransferase, and citrate synthase, with 1.3- to 10-fold increased Km values and 2.5- to 11-fold decreases in Vmax. The combined results indicate that the geminal dimethyl groups of CoA have modest effects on function and minimal effects on conformation.     

Identification of the active site residues in ATP‐citrate lyase's carboxy‐terminal portion

ATP‐citrate lyase (ACLY) catalyzes production of acetyl‐CoA and oxaloacetate from CoA and citrate using ATP. In humans, this cytoplasmic enzyme connects energy metabolism from carbohydrates to the production of lipids. In certain bacteria, ACLY is used to fix carbon in the reductive tricarboxylic acid cycle. The carboxy(C)‐terminal portion of ACLY shows sequence similarity to citrate synthase of the tricarboxylic acid cycle. To investigate the roles of residues of ACLY equivalent to active site residues of citrate synthase, these residues in ACLY from Chlorobium limicola were mutated, and the proteins were investigated using kinetics assays and biophysical techniques. To obtain the crystal structure of the C‐terminal portion of ACLY, full‐length C. limicola ACLY was cleaved, first non‐specifically with chymotrypsin and subsequently with Tobacco Etch Virus protease. Crystals of the C‐terminal portion diffracted to high resolution, providing structures that show the positions of active site residues and how ACLY tetramerizes.     

Activation of acyl condensation reaction of monomeric 6-hydroxymellein synthase,a multifunctional polyketide biosynthetic enzyme,by free coenzyme A

6-Hydroxymellein (6HM) synthase is a multifunctional polyketide enzyme induced in carrot cells, whose fully active homodimer catalyzes condensation of acyl-CoAs and the NADPH-dependent ketoreduction of the enzyme-bound intermediate. 6HM-forming activity of the synthase was markedly decreased when the reaction mixture pH was adjusted from 7.5 to 6.0. However, under these slightly acidic conditions, the acyl condensation catalyzed by the dissociated monomer enzyme was appreciably stimulated by addition of free coenzyme A (CoA). In contrast, the condensation reaction at pH 6.0 was significantly inhibited in the presence of CoA when the reaction was carried out with the NADPH-omitted dimer synthase. Among the kinetic parameters of the acyl condensation, velocity of the monomer-catalyzing reaction at the acidic pH was appreciably increased upon addition of CoA while K(m)s did not show any significant change in the presence and absence of the compound. These results suggest that CoA associates with a specific site in the dissociated monomeric form of 6HM synthase, and the velocity of the acyl condensation reaction catalyzed by the CoA-synthase complex appreciably increases in acidic conditions.     

Carboxymethylproline synthase from Pectobacterium carotorova: a multifaceted member of the crotonase superfamily

The simplest carbapenem antibiotic, (5R)-carbapen-2-em-3-carboxylic acid, is biosynthesized from primary metabolites in Pectobacterium carotorova by the action of three enzymes, carboxymethylproline synthase (hereafter named CarB), carbapenam synthetase, and carbapenem synthase. CarB, a member of the crotonase superfamily, catalyzes the formation of (2S,5S)-5-carboxymethylproline from malonyl-CoA and l-pyrroline-5-carboxylate. In this study we show that, in addition, CarB catalyzes the independent decarboxylation of malonyl-CoA and methylmalonyl-CoA and the hydrolysis of CoA esters such as acetyl-CoA and propionyl-CoA. The steady-state rate constants for these reactions are reported. We have identified the intermediates in the CarB reactions with l-pyrroline-5-carboxylate and malonyl-CoA or methylmalonyl-CoA as the CoA esters of (2S,5S)-5-carboxymethylproline and (2S,5S)-6-methyl-5-carboxymethylproline, respectively. The data provided indicate that these intermediates partition between completing turnover and dissociating from the enzyme. On the basis of the steady-state rate constants measured for the CarB-catalyzed hydrolysis of synthetic (2S,5S)-5-carboxymethylprolyl-CoA and for the CarB reaction with malonyl-CoA and l-pyrroline-5-carboxylate, we have calculated the rate constants for each step of these reactions. The results identify CarB as a particularly interesting member of the crotonase superfamily that combines in one net reaction three activities of this superfamily, decarboxylation, C-C bond formation, and CoA ester hydrolysis.     

The vanadium- and molybdenum-containing nitrogenases of Azotobacter chroococcum. Comparison of mid-point potentials and kinetics of reduction by sodium dithionite of the iron proteins with bound magnesium adenosine 5''-diphosphate.

The mid-point potentials of the Fe protein components (Ac2 and Ac2* respectively) of the Mo nitrogenase and V nitrogenase from Azotobacter chroococcum were determined in the presence of MgADP to be -450 mV (NHE) [Ac2(MgADP)2-Ac2*ox.(MgADP)2 couple] and -463 mV (NHE) [Ac2* (MgADP)2-Ac2*ox.(ADP)2 couple] at 23 degrees C at pH 7.2. These values are consistent with a flavodoxin characterized by Deistung & Thorneley [(1986) Biochem. J. 239, 69-75] with Em = -522 mV (NHE) being an effective electron donor to both the Mo nitrogenase and the V nitrogenase in vivo. Ac2*ox.(MgADP)2 and Ac2*ox.(MgADP)2 were reduced by SO2.- (formed by the predissociation of dithionite ion, S2O4(2-)) at similar rates, k = 4.7 X 10(6) +/- 0.5 X 10(6) M-1.s-1 and 3.2 X 10(6) +/- 0.2 X 10(6) M-1.s-1 respectively, indicating structural homology at the electron-transfer site associated with the [4Fe-4S] centre in these proteins.     

Kinetic study of the action of bovine chymosin and pepsin A on bovine kappa-casein

A study was carried out to determine the Michaelian parameters relative to the action of chymosin and pepsin A on bond Phe105-Met106 of bovine kappa0-casein (carbohydrate-free fraction in micellar state). The reaction was performed in citrate buffer, pH 6.2, at 30 degrees C. The reaction mixture was analysed by reverse phase HPLC. Dosages of peptide 106-169 (caseino macropeptide) at different reaction times from recordings of its absorbance at 220 nm gave the initial rates of reaction at each substrate concentration. From these values the following parameters were determined: kcat = 68.5 s-1, Km = 0.048 mM, kcat/Km = 1,413 mM-1 s-1 for chymosin, and kcat = 45 s-1, Km = 0.018 mM, kcat/Km = 2,439 mM-1 s-1 for pepsin A. For chymosin they are similar to those obtained previously in dimethyl glutarate buffer, pH 6.6, at 30 degrees C, using fragment 98-111 of kappa-casein as substrate. It can thus be concluded that neither the micellar state nor the presence of the whole peptide chain of kappa-casein (our conditions) significantly affect the action of chymosin on fragment 98-111, which seems to contain all information that makes bond 105-106 highly sensitive to chymosin. For pepsin A, only the information contained in fragment 103-108 appears to be required.     

Activation/dephosphorylation of muscle glycogen synthase phosphorylated by phosphorylase kinase

1. Glycogen synthase from rabbit skeletal muscle was phosphorylated by phosphorylase kinase to yield synthase b2. 2. Dephosphorylation and activation of synthase b2 by the catalytic subunits of protein phosphatase-1 (PP-1c) and protein phosphatase-2A (PP-2Ac) was studied. The apparent Km of PP-1c and PP-2Ac were 3.3 microM and 6.2 microM, respectively. The apparent Vmax of PP-1c was about two times larger than that of PP-2Ac. 3. Ligands with phosphate moiety (AMP, glucose-6-P at high concentration) caused an inhibition in dephosphorylation by both phosphatases. Spermine inhibited the dephosphorylation by PP-1c and stimulated the action of PP-2Ac. Therefore it can be employed to distinguish the phosphatases using synthase b2 as substrate.     

Kinetic and chemical mechanism of malate synthase from Mycobacterium tuberculosis

Malate synthase catalyzes the Claisen-like condensation of acetyl-coenzyme A (AcCoA) and glyoxylate in the glyoxylate shunt of the citric acid cycle. The Mycobacterium tuberculosis malate synthase G gene, glcB, was cloned, and the N-terminal His(6)-tagged 80 kDa protein was expressed in soluble form and purified by metal affinity chromatography. A chromogenic 4,4'-dithiodipyridine assay did not yield linear kinetics, but the generation of an active site-directed mutant, C619S, gave an active enzyme and linear kinetics. The resulting mutant exhibited kinetics comparable to those of the wild type and was used for the full kinetic analysis. Initial velocity studies were intersecting, suggesting a sequential mechanism, which was confirmed by product and dead-end inhibition. The inhibition studies delineated the ordered binding of glyoxylate followed by AcCoA and the ordered release of CoA followed by malate. The pH dependencies of k(cat) and k(cat)/K(gly) are both bell-shaped, and catalysis depends on a general base (pK = 5.3) and a general acid (pK = 9.2). Primary kinetic isotope effects determined using [C(2)H(3)-methyl]acetyl-CoA suggested that proton removal and carbon-carbon bond formation were partially rate-limiting. Solvent kinetic isotope effects on k(cat) suggested the hydrolysis of the malyl-CoA intermediate was also partially rate-limiting. Multiple kinetic isotope effects, utilizing D(2)O and [C(2)H(3)-methyl]acetyl-CoA, confirmed a stepwise mechanism in which the step exhibiting primary kinetic isotope effects precedes the step exhibiting the solvent isotope effects. We combined the kinetic data and the pH dependence of the kinetic parameters with existing structural and mutagenesis data to propose a chemical mechanism for malate synthase from M. tuberculosis.     

Multifunctionality of Campylobacter jejuni sialyltransferase CstII: characterization of GD3/GT3 oligosaccharide synthase, GD3 oligosaccharide sialidase, and trans-sialidase activities

CstII from bacterium Campylobacter jejuni strain OH4384 has been previously characterized as a bifunctional sialyltransferase having both alpha2,3-sialyltransferase (GM3 oligosaccharide synthase) and alpha2,8-sialyltransferase (GD3 oligosaccharide synthase) activities which catalyze the transfer of N-acetylneuraminic acid (Neu5Ac) from cytidine 5'-monophosphate (CMP)-Neu5Ac to C-3' of the galactose in lactose and to C-8 of the Neu5Ac in 3'-sialyllactose, respectively (Gilbert M, Karwaski MF, Bernatchez S, Young NM, Taboada E, Michniewicz J, Cunningham AM, Wakarchuk WW. 2002. The genetic bases for the variation in the lipo-oligosaccharide of the mucosal pathogen, Campylobacter jejuni. Biosynthesis of sialylated ganglioside mimics in the core oligosaccharide. J Biol Chem. 277:327-337). We report here the characterization of a truncated CstII mutant (CstIIDelta32(I53S)) cloned from a synthetic gene whose codons are optimized for an Escherichia coli expression system. In addition to the alpha2,3- and alpha2,8-sialyltransferase activities reported before for the synthesis of GM3- and GD3-type oligosaccharides, respectively, the CstIIDelta32(I53S) has alpha2,8-sialyltransferase (GT3 oligosaccharide synthase) activity for the synthesis of GT3 oligosaccharide. It also has alpha2,8-sialidase (GD3 oligosaccharide sialidase) activity that catalyzes the specific cleavage of the alpha2,8-sialyl linkage of GD3-type oligosaccharides and alpha2,8-trans-sialidase (GD3 oligosaccharide trans-sialidase) activity that catalyzes the transfer of a sialic acid from a GD3 oligosaccharide to a different GM3 oligosaccharide (3'-sialyllactoside). The donor substrate specificity study of the CstIIDelta32(I53S) GD3 oligosaccharide synthase activity indicates that the enzyme is flexible in using different CMP-activated sialic acids and their analogs for the synthesis of GD3 oligosaccharides containing natural and nonnatural modifications at the terminal sialic acid.     

Properties of the thioesterase component obtained by limited trypsinization of the fatty acid synthetase multienzyme complex.

The fatty acid synthetase from lactating rat mammary gland is shown to consist of two polyfunctional polypeptides of similar molecular weight (about 220,000); a 4'-phosphopantetheine residue is covalently bound to one, or both subunits. Limited trypsinization of the fatty acid synthetase releases on enzymatically active thioesterase component which has been purified and its properties studied. The thioesterase sediments in the ultracentrifuge as a single component of molecular weight 32,000; its sedimentation coefficient is 2.9 x 10-(13) s its diffusion coefficient 5.0 x 10-(7) cm2 s-(1). The thioesterase also elutes from a column of Sephadex G-75 as a single, symmetrical peak of constant specific activity. However, electrophoresis of the denatured thioesterase in the presence of sodium dodecyl sulfate reveals that the enzyme has been partially nicked during isolation. The kinetic data of the enzyme reaction were studied using palmityl-CoA as a model substrate. Solvent pH was found to affect both Vmax and Km (Km = 0.5 micron at pH 6.6, 2.5 micron at pH 8.0) wereas solvent ionic strength affected Vmax but no Km. The thioesterases from the fatty acid synthetases of rat liver and lactating mammary gland have identical physical properties, identical amino acid compositions, and are immunologically indistinguishable. Both thioesterases hydrolyze long chain, in preference to short chain, thioesters of CoA, an observation consistent with their role in regulation of the chain-terminating step in fatty acid synthesis by the parent multienzyme complexes.     

Purification and some properties of the citrate synthase from a marine Pseudomonas.

Citrate synthase (citrate-oxaloacetate lyase (CoA acetylating), EC 4.1.3.7) has been purified to electrophoretic homogeneity from a marine Pseudomonas. The enzyme was made up of identical subunits, with a molecular wieght of about 53 000, as determined by sodium dodecyl sulphate - polyacrylamide gel electrophoresis. The native enzyme (citrate synthase II, CS II) could be dissociated by dialysis against 20 mM phosphate (Pi), pH 7; the enzyme thus obtained (citrate synthase I, CS I) was still active, but presented different molecular weight and kinetic and regulatory properties. CS II was activated by adenosine monophosphate (AMP), Pi, and KCl, and inhibited by reduced nicotinamide adenine dinucleotide (NADH), being apparently insensitive to adenosine triphosphate (ATP) and adenosine diphosphate (ADP). The inhibition by NADH was completely counteracted by 0.1 mM AMP, but not by 50 mM Pi or 0.1 M KCl. The activation by KCl and Pi, or by KCl and AMP was nearly additive, whereas that by AMP and Pi was not. The activators acted essentially by increasing Vmax, although they also caused a decrease in the Km values. CS I was inhibited by ATP, ADP, AMP, and KCl, and was insensitive to NADH. CS I could be reassociated after elimination of Pi by dialysis, regaining the higher molecular weight and the activation by AMP characteristic of CS II.     

Subunit equilibria of porcine heart citrate synthase. Effects of enzyme concentration, pH, and substrates

Porcine heart citrate synthase, a dimeric protein of Mr = 100,000 composed of two identical subunits, is shown to undergo a monomer-dimer equilibrium. The extent of dimerization is found to be dependent on the concentration of citrate synthase, pH, ionic strength, and the specific buffer system employed. Oxaloacetate and citrate, substrates for the forward and reverse reaction catalyzed by citrate synthase, affect dimerization at concentrations of the protein which exists as monomer in their absence. The dissociation of citrate synthase dimers has been demonstrated utilizing the techniques of gel permeation chromatography, fluorescence polarization, fluorescence energy transfer, and heat denaturation. Earlier studies of citrate synthase quarternary structure found the protein to be nondissociable except under denaturing conditions or extensive modification; however, most former studies were performed at relatively high protein concentration, ionic strength, and pH, conditions which stabilize the dimer. In light of recent evidence derived from x-ray crystallographic studies showing amino acid residues from one subunit contributing to the citrate and CoA binding sites of the other, the dissociation into monomers would be expected to have profound effects on citrate synthase activity and regulation, as well as overall tricarboxylic acid cycle activity.     

Mitochondrial malate dehydrogenase and malic enzyme of a filarial worm Setaria digitata: some properties and effects of drugs and herbal extracts.

Mitochondrial malate dehydrogenase (mMDH) and malic enzyme (mME) of a filarial worm Setaria digitata were studied. mMDH exhibited the highest activities in the oxidation and reduction reactions at pH 9.5 and pH 6.2, respectively, while mME did so in the malate decarboxylation reaction at pH 6.8. mME showed no detectable activity on the pyruvate carboxylation direction. The Km values for malate (1.7 mM) and oxaloacetate (0.17 mM) and the ratio of Vmax oxidation: Vmax reduction (2.73) tend to favor the oxaloacetate reduction by mMDH. mME showed a relatively high Km value of 8.3 mM, for malate decarboxylation. A drug, diethylcarbamazine citrate (DEC-C), did not change appreciably the activity of either mMDH or mME, while filarin (a drug of herbal origin) effectively inhibited mMDH. The leaf extracts of Ocimum sanctum, Lawsonia inermis and Calotropis gigantea and leaf and flower extracts of Azadirachta indica were, however, found to inhibit both mMDH and mME.     

Detection of a transient enzyme-steroid complex during active-site-directed irreversible inhibition of 3-oxo-delta 5-steroid isomerase

The reaction of the active-site-directed irreversible inhibitor (17S)-spiro[estra-1,3,5(10),6,8-pentaene-17,2'-oxiran]-3-ol (5 beta) with 3-oxo-delta 5-steroid isomerase has been monitored by repetitive scanning ultraviolet spectroscopy of a solution of 5 beta plus isomerase against a blank containing only 5 beta. Upon initial mixing of 5 beta with the isomerase an absorbance maximum at ca. 250 nm appears. With time, this peak decreases and is replaced with a new peak near 280 nm. These results directly demonstrate the existence of a transient enzyme-steroid intermediate in the inactivation reaction. The ultraviolet spectrum suggests that the steroid in the transient complex resembles the ionized phenol, while the phenolic group in the irreversibly bound complex is un-ionized. These spectral studies support our previous proposal that there are two enzyme-steroid complexes that are related by a 180 degree rotation about an axis perpendicular to the plane of the steroid nucleus. This hypothesis offers an explanation for the reaction of 17 beta-oxiranes with the same residue (Asp-38) that is thought to be involved in the catalytic mechanism. Two new oxiranes, (17S)-spiro[estra-1,3,5(10)-triene-17,2'-oxiran]-3 beta-ol (6 beta) and (17S)-spiro[5 alpha-androstane-17,2'-oxiran]-3-one (8 beta), were also found to be potent active-site-directed irreversible inhibitors of the isomerase (k3/KI = 31 M-1 s-1 and 340 M-1 s-1, respectively). The relationship of these results to the nature of the active site of the isomerase is discussed.     

Low- and high-affinity transport systems for citric acid in the yeast Candida utilis.

Citric acid-grown cells of the yeast Candida utilis induced two transport systems for citric acid, presumably a proton symport and a facilitated diffusion system for the charged and the undissociated forms of the acid, respectively. Both systems could be observed simultaneously when the transport was measured at 25 degrees C with labelled citric acid at pH 3.5 with the following kinetic parameters: for the low-affinity system, Vmax, 1.14 nmol of undissociated citric acid s-1 mg (dry weight) of cells-1, and Km, 0.59 mM undissociated acid; for the high-affinity system, Vmax, 0.38 nmol of citrate s-1 mg (dry weight) of cells-1, and Km, 0.056 mM citrate. At high pH values (above 5.0), the low-affinity system was absent or not measurable. The two transport systems exhibited different substrate specificities. Isocitric acid was a competitive inhibitor of citric acid for the high-affinity system, suggesting that these tricarboxylic acids used the same transport system, while aconitic, tricarballylic, trimesic, and hemimellitic acids were not competitive inhibitors. With respect to the low-affinity system, isocitric acid, L-lactic acid, and L-malic acid were competitive inhibitors, suggesting that all of these mono-, di-, and tricarboxylic acids used the same low-affinity transport system. The two transport systems were repressed by glucose, and as a consequence diauxic growth was observed. Both systems were inducible, and not only citric acid but also lactic acid and malic acid may induce those transport systems. The induction of both systems was not dependent on the relative concentration of the anionic form(s) and of undissociated citric acid in the culture medium.(ABSTRACT TRUNCATED AT 250 WORDS)     

Low- and high-affinity transport systems for citric acid in the yeast Candida utilis.

Citric acid-grown cells of the yeast Candida utilis induced two transport systems for citric acid, presumably a proton symport and a facilitated diffusion system for the charged and the undissociated forms of the acid, respectively. Both systems could be observed simultaneously when the transport was measured at 25 degrees C with labelled citric acid at pH 3.5 with the following kinetic parameters: for the low-affinity system, Vmax, 1.14 nmol of undissociated citric acid s-1 mg (dry weight) of cells-1, and Km, 0.59 mM undissociated acid; for the high-affinity system, Vmax, 0.38 nmol of citrate s-1 mg (dry weight) of cells-1, and Km, 0.056 mM citrate. At high pH values (above 5.0), the low-affinity system was absent or not measurable. The two transport systems exhibited different substrate specificities. Isocitric acid was a competitive inhibitor of citric acid for the high-affinity system, suggesting that these tricarboxylic acids used the same transport system, while aconitic, tricarballylic, trimesic, and hemimellitic acids were not competitive inhibitors. With respect to the low-affinity system, isocitric acid, L-lactic acid, and L-malic acid were competitive inhibitors, suggesting that all of these mono-, di-, and tricarboxylic acids used the same low-affinity transport system. The two transport systems were repressed by glucose, and as a consequence diauxic growth was observed. Both systems were inducible, and not only citric acid but also lactic acid and malic acid may induce those transport systems. The induction of both systems was not dependent on the relative concentration of the anionic form(s) and of undissociated citric acid in the culture medium.(ABSTRACT TRUNCATED AT 250 WORDS)     

Expression of spinach glycolate oxidase in Saccharomyces cerevisiae: purification and characterization.

Glycolate oxidase from spinach has been expressed in Saccharomyces cerevisiae. The active enzyme was purified to near-homogeneity (purification factor approximately 1400-fold) by means of hydroxyapatite and anion-exchange chromatography. The purified glycolate oxidase is nonfluorescent and has absorbance peaks at 448 (epsilon = 9200 M-1 cm-1) and 346 nm in 0.1 M phosphate buffer, pH 8.3. The large bathochromic shift of the near-UV band indicates that the N(3) position is deprotonated at pH 8.3. A pH titration revealed that the pK of the N(3) is shifted from 10.3 in free flavin to 6.4 in glycolate oxidase. Glycolate oxidase is competitively inhibited by oxalate with a Kd of 0.24 mM at 4 degrees C in 0.1 M phosphate buffer, pH 8.3. Three pieces of evidence demonstrate that glycolate oxidase stabilizes a negative charge at the N(1)-C(2 = O) locus: the enzyme forms a tight sulfite complex with a Kd of 2.7 x 10(-7) M and stabilizes the anionic flavosemiquinone and the benzoquinoid form of 8-mercapto-FMN. Steady-state analysis at pH 8.3, 4 degrees C, yielded a Km = 1 x 10(-3) M for glycolate and Km = 2.1 x 10(-4) M for oxygen. The turnover number has been determined to be 20 s-1. Stopped-flow studies of the reductive (k = 25 s-1) and oxidative (k = 8.5 x 10(4) M-1 s-1) half-reactions have identified the reduction of glycolate oxidase to be the rate-limiting step.