The effects of calcium ions and adenine nucleotides on the activity of pig heart 2-oxoglutarate dehydrogenase complex.

1. The effects of Ca2+ (mainly by using EGTA buffers), pH, ATP and ADP on the activity of the 2-oxoglutarate dehydrogenase complex from pig heart were explored. 2. Ca2+ (about 30 micrometer) resulted in a decrease in the apparent Km for 2-oxoglutarate from 2.1 to 0.16 mM (at pH 7) without altering the maximal velocity. At 0.1 mM-oxoglutarate there was a 4--5-fold activation by Ca2+, with an apparent Km for Ca2+ of 1.2 micrometer. A similar activation was also observed with Sr2+ (Km 15.1 micrometer), but not wised markedly from pH 7.4 TO 6.6. The effects of Ca2+ remained evident over this pH range. 4. In the presence of Mg2+, ATP resulted in a marked increase in the apparent Km for oxoglutarate, whereas ADP greatly decreased thisp arameter. The concentrations of adenine nucleotide required for half-maximal effects were about 10 micrometer in each case. 5. The effects of the adenine nucleotides and Ca2+ on the apparent Km for oxoglutarate appeared to be essentially independent of each other, reversible, and demonstrable in the presence of end product inhibition by NADH and obtained. 6. Effects similar to those described above were also observed on the activity of 2-oxoglutarate dehydrogenase from rat heart and brown adipose tissue. 7. We discuss the mechanisms controlling this enzyme's activity and compare these regulatory features with those of NAD-isocitrate dehydrogenase and the pyruvate dehydrogenase system, which are also sensitive to Ca2+ and adenine nucleotides.     

A kinetic study of the alpha-keto acid dehydrogenase complexes from pig heart mitochondria.

The kinetic mechanisms of the 2-oxoglutarate and pyruvate dehydrogenease complexes from pig heart mitochondria were studied at pH 7.5 and 25 degrees. A three-site ping-pong mechanism for the actin of both complexes was proposed on the basis of the parallel lines obtained when 1/v was plotted against 2-oxoglutarate or pyruvate concentration for various levels of CoA and a level of NAD+ near its Michaelis constant value. Rate equations were derived from the proposed mechanism. Michaelis constants for the reactants of the 2-oxoglutarate dehydrogenase complex reaction are: 2-oxoglutarate, 0.220 mM; CoA, 0.025 mM; NAD+, 0.050 mM. Those of the pyruvate dehydrogenase complex are: pyruvate, 0.015 mM; CoA, 0.021 mM; NAD+, 0.079 mM. Product inhibition studies showed that succinyl-CoA or acetyl-CoA was competitive with respect to CoA, and NADH was competitive with respect to NAD+ in both overall reactions, and that succinyl-CoA or acetyl-CoA and NADH were uncompetitive with respect to 2-oxoglutarate or pyruvate, respectively. However, noncompetitive (rather than uncompetitive) inhibition patterns were observed for succinyl-CoA or acetyl-CoA versus NAD+ and for NADH versus CoA. These results are consistent with the proposed mechanisms.     

Ox liver glutamate dehydrogenase. The role of lysine-126 reappraised in the light of studies of inhibition and inactivation by pyridoxal 5''-phosphate.

The time-course of inactivation of bovine liver glutamate dehydrogenase by pyridoxal 5'-phosphate was studied in the presence of varied amounts of 2-oxoglutarate or NADH. Pseudo-first-order analysis reveals that the protection by both these compounds is competitive with respect to the chemical modifier. The competition is only partial, however: saturation with either NADH or 2-oxoglutarate decreases the rate constant for inactivation to a finite minimum and not to zero. Similarly, the plot of activity at equilibrium as a function of the concentration of the protecting substrate or coenzyme reveals that neither NADH nor 2-oxoglutarate protects completely against inactivation. In initial-rate experiments, pyridoxal 5'-phosphate, used as an instantaneous inhibitor rather than a long-term inactivator, displayed non-competitive inhibition with respect to both 2-oxoglutarate and NADH. These results clearly indicate that, although there is mutual hindrance between the binding to the enzyme of pyridoxal 5'-phosphate, on the one hand, and 2-oxoglutarate or NADH on the other, binding is not mutually exclusive. These findings are discussed in terms of the two-step mechanism for inactivation by pyridoxal 5'-phosphate. It is concluded that lysine-126 cannot be solely responsible for binding either the substrate or the coenzyme, but could be essential for the catalytic step.     

Studies on a mutant form of Escherichia coli citrate synthase desensitised to allosteric effectors.

Naturally occurring citrate synthases fall into distinct molecular and catalytic types. Gram-negative bacteria produce a 'large' enzyme, allosterically inhibited by NADH and, in the facultative anaerobes such as Escherichia coli, also by 2-oxoglutarate. On the other hand, Gram-positive bacteria and all eukaryotes produce a 'small' citrate synthase which is insensitive to these metabolites. As a complement to structure-function studies we have explored the possibility of genetically altering one type of citrate synthase to the other. By mutagenesis and suitable selection we have succeeded in isolating a mutant of E. coli whose citrate synthase is both 'small' and insensitive to NADH and 2-oxoglutarate. Some characteristics of the enzyme are described. Such mutant enzymes offer a novel approach to the study of citrate synthase, its regulation and its natural diversity.     

Kinetic mechanism of NADH-dependent glutamate synthase from lupin nodules.

From initial-rate studies, a partially random kinetic mechanism has been deduced for NADH-dependent glutamate synthase from lupin nodules. The mechanism involves compulsory binding of NADH as first substrate, followed by random-order binding of glutamine and 2-oxoglutarate. Patterns of inhibition by glutamate substantiate the mechanism. Dithionite was incapable of acting as an alternative reducing substrate although it is known to reduce the flavine groups of the enzyme. The implications of these results are discussed. Published rate equations for this type of mechanism were found to be unsatisfactory for this enzyme and suitable new equations are produced. These equations should have general application where the obligatory first substrate binds very tightly.     

Evidence for allosteric NADH regulation of Acinetobacter alpha-oxoglutarate dehydrogenase from multiple-inhibition studies.

Previous studies have suggested that the E1 component (α-oxoglutarate dehydrogenase) of the α-oxoglutarate dehydrogenase enzyme complex from $\text{Acinetobacter lwoffi}$ is inhibited by the end-product NADH. We have now carried out multiple-inhibition studies in the simultaneous presence of NADH and α-oxoadipate, both competitive inhibitors with respect to α-oxoglutarate. The results indicate that NADH acts at an allosteric site within the multi-enzyme complex.     

Mechanistic studies on Azospirillum brasilense glutamate synthase.

The reaction mechanism of Azospirillum brasilense glutamate synthase has been investigated by several approaches. 15N nuclear magnetic resonance studies demonstrate that the amide nitrogen of glutamine is reductively transferred to 2-oxoglutarate in an irreversible manner with no release of the transferred ammonia group into the medium. Identical results were obtained using thio-NADPH and acetylpyridine-NADPH, which are shown to be less efficient substrates of the enzyme than NADPH. Similarly, no exchange of the ammonia group being transferred with exogenous ammonium ion was observed during catalysis. The glutamate formed as the product of the iminoglutarate reduction was determined to be in the L configuration. The enzyme was also found to catalyze, under anaerobic conditions, the exchange of the 4proS H of NADPH with solvent both in the absence and in the presence of 2-oxoglutarate and glutamine. The reductive half-reaction is therefore a reversible segment of the overall irreversible amidotransferase reaction. 15N NMR studies also showed that the enzyme does not catalyze glutamate dehydrogenase/oxidase reactions or any observable glutaminase activity under neutral (pH 7.5) conditions. Glutaminase activity was also not observable with the reduced enzyme alone or in the presence of D-glutamate (a competitive inhibitor of glutamate synthase with respect to 2-oxoglutarate, with a Ki of about 11 microM) or with the oxidized enzyme in the presence of 2-oxoglutarate, D-glutamate, or NADP+. These data confirm species-dependent differences of A. brasilense glutamate synthase with respect to the enzyme from other sources.     

Time-dependent inactivation of chick-embryo prolyl 4-hydroxylase by coumalic acid. Evidence for a syncatalytic mechanism.

From the structure-activity relationships of known competitive inhibitors, coumalic acid (2-oxo-1,2H-pyran-5-carboxylic acid) was deduced to be a potential syncatalytic inhibitor for chick-embryo prolyl 4-hydroxylase. The compound caused time-dependent inactivation, the reaction rate being first-order. The inactivation constant was 0.094 min-1, the Ki 17 mM and the bimolecular rate constant 0.09 M-1 X S-1. Human prolyl 4-hydroxylase and chick embryo lysyl hydroxylase were also inactivated, though to a lesser extent. Inactivation could be prevented by adding high concentrations of 2-oxoglutarate or its competitive analogues to the reaction mixture. In Lineweaver-Burk kinetics, coumalic acid displayed S-parabolic competitive inhibition with respect to 2-oxoglutarate. The inactivation reaction had cofactor requirements similar to those for the decarboxylation of 2-oxoglutarate. Enzymic activity was partially preserved in the absence of iron, but the rescue was incomplete, owing to decreased stability of the enzyme under this condition. Coumalic acid also decreased the electrophoretic mobility of the alpha-subunit, but the beta-subunit was not affected. Prolonged incubation of coumalic acid above pH 6.8 led to loss of its inactivating potency, owing to hydrolysis. It is concluded that the inactivation of prolyl 4-hydroxylase by coumalic acid is due to a syncatalytic mechanism. The data also suggest that the 2-oxoglutarate-binding site of the enzyme is located within the alpha-subunit.     

Some kinetic and other properties of the isoenzymes of aspartate aminotransferase isolated from sheep liver.

A method for the purification of mitochondrial isoenzyme of sheep liver aspartate aminotransferase (EC 2.6.1.1) is described. The final preparation is homogeneous by ultracentrifuge analyses and polyacrylamide-gel electrophoresis and has a high specific activity (182 units/mg). The molecular weight determined by sedimentation equilibrium is 87,100 +/- 680. The amino acid composition is presented; it is similar to that of other mitochondrial isoenzymes, but with a higher content of tyrosine and threonine. Subforms have been detected. On isoelectric focusing a broad band was obtained, with pI 9.14. The properties of the mitochondrial aspartate aminotransferase are compared with those of the cytoplasmic isoenzyme. The Km for L-aspartate and 2-oxoglutarate for the cytoplasmic enzyme were 2.96 +/- 0.20 mM and 0.093 +/- 0.010 mM respectively; the corresponding values for the mitochondrial form were 0.40 +/- 0.12 mM and 0.98 +/- 0.14 mM. Cytoplasmic aspartate aminotransferase showed substrate inhibition by concentrations of 2-oxoglutarate above 0.25 mM in the presence of aspartate up to 2mM. The mitochondrial isoenzyme was not inhibited in this way. Pi at pH 7.4 inhibited cytoplasmic holoenzyme activity by up to about 60% and mitochondrial holoenzyme activity up to 40%. The apparent dissociation constants for pyridoxal 5'-phosphate were 0.23 micrometer (cytoplasmic) and 0.062 micrometer (mitochondrial) and for pyridoxamine 5'-phosphate they were 70 micrometer (cytoplasmic) and 40 micrometer (mitochondrial). Pi competitively inhibited coenzyme binding to the apoenzymes; the inhibition constants at 37 degree C were 32 micrometer for the cytoplasmic isoenzyme and 19.5 micrometer for the mitochondrial form.     

A product-inhibition study of bovine liver glutamate dehydrogenase.

1. Initial rates of oxidative deamination of L-glutamate with NAD+ as coenzyme, and of reductive aminiation of 2-oxoglutarate with NADH as coenzyme, catalysed by bovine liver glutamate dehydrogenase were measured in 0.111 M-sodium phosphate buffer, pH 7, at 25 degrees C, in the absence and presence of product inhibitors. All 12 possible combinations of variable substrate and product inhibitor were used. 2. Strict competition was observed between NAD+ and NADH, and between glutamate and 2-oxoglutarate. All other inhibition patterns were clearly non-competitive, except for inhibition by NH4+ with NAD+ as variable substrate. Here the extrapolation did not permit a clear distinction between competitive and non-competitive inhibition. 3. Mutually non-competitive behaviour between glutamate and NH4+ indicates that these substrates can be bound at the active site simultaneously. 4. Primary Lineweaver-Burk plots and derived secondary plots of slopes and intercepts against inhibitor concentration were linear, with one exception: with 2-oxoglutarate as variable substrate, the replot of primary intercepts against inhibitory NAD+ concentration was curved. 5. Separate Ki values were evaluated for the effect of each product inhibitor on the individual terms in the reciprocal initial-rate equations. With this information it is possible to calculate rates for any combination of substrate concentrations within the experimental range with any concentration of a single product inhibitor. 6. The inhibition patterns are consistent with neither a simple compulsory-order mechanism nor a rapid-equilibrium random-order mechanism without modification. They can, however, be reconciled with either type of mechanism by postulating appropirate abortive complexes. Of the two compulsory sequences that have been proposed, one, that in which the order of binding is NADH, NH4+, 2-oxoglutarate, requires an implausible pattern of abortive complex-formation to account for the results. 7. On the basis of a rapid-equilibrium random-order mechanism, dissociation constants can be calculated from the Ki values. Where these can be compared with independent estimates from the kinetics of the uninhibited reaction or from direct measurements of substrate binding, the agreement is reasonable good. On balance, therefore, the results provide further support for the rapid-equilibrium random-order mechanism under these conditions.     

Properties of apoglutamate synthase and comparison with glutamate dehydrogenase.

Glutamate synthase from Escherichia coli K-12 exhibits NH3-dependent activity. NH3-dependent activity is increased approximately 5-fold in apoglutamate synthase lacking flavin and non-heme iron. Whereas glutamine plus 2-oxoglutarate have the capacity to reoxidize the chemically reduced flavoenzyme, no such reoxidation is obtained with 2-oxoglutarate plus NH3. These results establish that the glutamine- and NH3-dependent syntheses of glutamate occur by different pathways of electron transfer from NADPH. The NH3-dependent activity of native and apoglutamate synthase exhibits similar catalytic properties. Some properties of apoglutamate synthase are similar to those of glutamate dehydrogenase. These properties include pH optima for synthesis and oxidative deamination of glutamate, inactivation by alkylating reagents and p-mercuribenzoate, an enhanced rate of inactivation by alkylating reagents and p-mercuribenzoate at low pH, 2-oxoglutarate protection against inactivation by p-mercuribenzoate, and reactivation of p-mercuribenzoate-treated enzyme by 2-mercaptoethanol. 2-Oxoglutarate protects against alkylation of glutamate synthase by iodo [1-14C]acetamide and reduces incorporation of methyl [1-14C]carboxamide into the small subunit of the enzyme.     

Partial reversal of the acetaldehyde and butyraldehyde oxidation reactions catalysed by aldehyde dehydrogenases from sheep liver.

In the presence of acetic anhydride or butyric anhydride, liver aldehyde dehydrogenases catalyse the oxidation of NADH at pH 7.0 and 25 degrees C. The maximum velocities and Michaelis constants for NADH at saturating anhydride concentrations are independent of which anhydride is used, the values being V'max. = 12 min-1 and Km for NADH = 9 micrometer for the mitochondrial enzyme and V'max = 25 min-1 and Km for NADH = 20 micrometer for the cytoplasmic enzyme. Substitution of [4A-2H]NADH for NADH resulted in 2-fold and 4-fold decreases in rate for the mitochondrial and cytoplasmic enzymes respectively.     

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.     

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.     

Purification and properties of a citrate synthase from Nitrosomonas sp. TK794 and a comparison with the enzymes of another nitrifying bacteria

Citrate(si)-synthase (citrate oxaloacetate-lyasem EC 4.1.3.7) was purified as an electrophoretically homogeneous protein from an ammonia-oxidizing chemoautotrophic bacterium, Nitrosomonas sp. TK794. The molecular mass of the native enzyme was estimated to be about 287 kDa by gel filtration, whereas SDS-PAGE produced one band with M_r values of 44.7 kDa, suggesting that the enzyme is a hexamer consisting of identical subunits. The isoelectric point of the enzyme was 5.0. The pH and temperature optima for citrate synthase (CS) activity was about 7.5–8.0 and 40°C, respectively. The citrate synthase was stable over a pH range of 6.0–8.5 and up to 40°C. The apparent K_m values for oxaloacetate and acetyl-CoA were about 11 μM and 247 μM, respectively. The activity of the citrate synthase was not inhibited by ATP, NADH or 2-oxoglutarate at 5mM, and was activated by potassium chloride at 0.1–100 mM. The N-terminal amino acid sequence of the enzyme protein was PPQDVATLSPGENKKTIELPILG.     

Factors affecting the activity of citrate synthase of Acetobacter xylinum and its possible regulatory role.

The citrate synthase activity of Acetobacter xylinum cells grown on glucose was the same as of cells grown on intermediates of the tricarboxylic acid cycle. The activity of citrate synthase in extracts is compatible with the overall rate of acetate oxidation in vivo. The enzyme was purified 47-fold from sonic extracts and its molecular weight was determined to be 280000 by gel filtration. It has an optimum activity at pH 8.4. Reaction rates with the purified enzyme were hyperbolic functions of both acetyl-CoA and oxaloacetate. The Km for acetyl-CoA is 18 mum and that for oxaloacetate 8.7 mum. The enzyme is inhibited by ATP according to classical kinetic patterns. This inhibition is competitive with respect to acetyl-CoA (Ki = 0.9 mM) and non-competitive with respect to oxaloacetate. It is not affected by changes in pH and ionic strength and is not relieved by an excess of Mg2+ ions. Unlike other Gram-negative bacteria, the A. xylinum enzyme is not inhibited by NADH, but is inhibited by high concentrations of NADPH. The activity of the enzyme varies with energy charge in a manner consistent with its role in energy metabolism. It is suggested that the flux through the tricarboxylic acid cycle in A. xylinum is regulated by modulation of citrate synthase activity in response to the energy state of the cells.     

Separation of two forms of glutamate synthase in leaves of tomato (Lycopersicon esculentum)

Two forms of glutamate synthase, one dependent on NAD(P)H, and the other on ferredoxin, have been completely separated by ionic exchange chromatography on DEAE cellulose. The NAD(P)H dependent enzyme was further purified by affinity chromatography with Blue Sepharose, showing Km values of 0.5 mM, 0.3 mM and 1.7 μM for glutamine, 2-oxoglutarate and NADH, respectively. Ferredoxin dependent enzyme was also purified to electrophoretic homogeneity; the Km values were 0.5 mM, 0.2 mM and 0.2 μM for glutamine, 2-oxoglutarate and ferredoxin, respectively. These results support the glutamine synthetase-glutamate synthase pathway for nitrogen assimilation.     

The evolution of glutamate synthase

DNA coding for the ferredoxin-dependent glutamate synthase (EC1.4.7.1) of spinach chloroplasts has been cloned and sequenced. It consists of 5015 bp and starts with the codon for the N-terminal cysteine of the mature protein. Ferredoxin-dependent glutamate synthase is one of the key enzymes in the early stages of ammonia assimilation in plants, algae and cyanobacteria. In addition to the ferredoxin-dependent enzyme, there are two other forms of glutamate synthase, one of which uses NADH as the electron donor and a second that uses NADPH. Although all three forms catalyze the reductive transamidation of the amido nitrogen from glutamine to 2-oxoglutarate to form two molecules of glutamate, ferredoxin-dependent glutamate synthases differ from the NADH and NADPH-dependent forms in subunit composition and amino acid sequence. The recent availability of sequence data for glutamate synthases from spinach and from two archael species has produced a clearer and more detailed picture of the evolution of this key enzyme in nitrogen metabolism and the origins of the two subunit/domain structure of the enzyme.     

Purification and characterization of flavone synthase I, a 2-oxoglutarate-dependent desaturase

Soluble flavone synthase I from illuminated parsley cells was purified to near homogeneity by a six-step procedure. A molecular mass of 48 +/- 2 kDa was determined by gel permeation chromatography and denaturing polyacrylamide gel electrophoresis. A single protein with an isoelectric point at pH 4.8 +/- 0.1 was detected on isoelectric focusing gels, which catalyzed the overall conversion of 2S-flavanones into the corresponding flavones in the presence of molecular oxygen, 2-oxoglutarate, ferrous ion, and ascorbate. Apparent Michaelis constants for 2S-naringenin, 2S-eriodictyol, and 2-oxoglutarate were determined as 5, 8, and 16 microM, respectively. (+)-Dihydrokaempferol and 2R-naringenin were not accepted as substrates. The enzyme was strongly inhibited by Cu2+ and Zn2+. Potent competitive inhibition with respect to 2-oxoglutarate was observed with 2,4-pyridinedicarboxylate (Ki = 1.8 microM). With crude extracts as well as with the purified enzyme neither the hypothetical intermediate 2-hydroxyflavanone nor a dehydratase activity capable of converting the chemically synthesized compound to flavone could be observed. Moreover, the introduction of the double bond into the substrate naringenin was not altered by addition of chemically synthesized 2-hydroxynaringenin into the reaction mixture. Therefore, 2-hydroxyflavanones are apparently not freely dissociable intermediates in the biosynthesis of flavones in parsley and are not capable of entering the active site of the enzyme to compete with the flavanone. It is postulated that flavone synthase I catalyzes double-bond formation by direct abstraction of vicinal hydrogen atoms at C-2 and C-3 of the substrate. Thus, flavone synthase I is a member of a novel subgroup within the 2-oxoglutarate-dependent dioxygenases that can be referred to as 2-oxoglutarate-dependent desaturases.     

Gram-positive type citrate synthase in the thermophilic green gliding bacterium Chloroflexus aurantiacus

Abstract Some properties of the citrate synthase from Chloroflexus aurantiacus have been examined in crude cell-free extracts and partially purified preparations. The enzyme had an approximate native molecular size of 140 000, was not inhibited by NADH or 2-oxoglutarate but was inhibited by ATP (about 50% at 5 mM). The K _m for acetyl CoA at pH 8.2 in the presence of 0.5 mM oxaloacetate was determined to be 25 μM.
These properties are characteristic of the 'small' size class of citrate synthases normally associated with gram-positive eubacteria, despite the fact that Chloroflexus stains gram-negatively.