New spectrophotometric and radiochemical assays for acetyl-CoA: arylamine N-acetyltransferase applicable to a variety of arylamines

Simple and sensitive spectrophotometric and radiochemical procedures are described for the assay of acetyl-CoA:arylamine N-acetyltransferase (NAT; EC 2.3.1.5), which catalyzes the reaction acetyl-CoA + arylamine----N-acetylated arylamine + CoASH. The methods are applicable to crude tissue homogenates and blood lysates. The spectrophotometric assay is characterized by two features: (i) NAT activity is measured by quantifying the disappearance of the arylamine substrate as reflected by decreasing Schiff's base formation with dimethylaminobenzaldehyde. (ii) During the enzymatic reaction, the inhibitory product CoASH is recycled by the system acetyl phosphate/phosphotransacetylase to the substrate acetyl-CoA. The radiochemical procedure depends on enzymatic synthesis of [3H]acetyl-CoA in the assay using [3H]acetate, ATP, CoASH, and acetyl-CoA synthetase. NAT activity is measured by quantifying N-[3H]acetylarylamine after separation from [3H]acetate by extraction. Product inhibition by CoASH is prevented in this system by the use of acetyl-CoA synthetase.     

Regulation of fatty acid beta-oxidation in rat heart mitochondria

In an attempt to elucidate the mechanism by which the rate of fatty acid oxidation is tuned to the energy demand of the heart, the effects of changing intramitochondrial ratios of [acetyl-CoA]/[CoASH] and [NADH]/[NAD+] on the rate of beta-oxidation were studied. When 10 mM L-carnitine was added to coupled rat heart mitochondria to lower the ratio of [acetyl-CoA]/[CoASH], the rate of palmitoylcarnitine beta-oxidation, as measured by the formation of acid-soluble products, was stimulated more than fourfold at state 4 respiration while beta-oxidation at state 3 respiration was hardly affected. Neither oxaloacetate nor acetoacetate, added to mitochondria to lower the [NADH]/[NAD+] ratio, stimulated beta-oxidation. Rates of respiration at states 3 and 4 were unchanged by additions of L-carnitine, oxaloacetate, or acetoacetate. Determinations of intramitochondrial ratios of [acetyl-CoA]/[CoASH] by high performance liquid chromatography yielded values close to 10 for palmitoylcarnitine-supported respiration at state 4 and 2.5 at state 3 respiration. Addition of 10 mM L-carnitine caused a dramatic decrease of these ratios to less than 0.2 at both respiration states. Studies with purified or partially purified enzymes revealed strong inhibitions of 3-ketoacyl-CoA thiolase by acetyl-CoA and of L-3-hydroxyacyl-CoA dehydrogenase by NADH. Moreover, the activity of 3-ketoacyl-CoA thiolase at concentrations of acetyl-CoA and CoASH prevailing at state 3 respiration was 4 times higher than its activity in the presence of acetyl-CoA and CoASH observed at state 4. Altogether, this study leads to the conclusion that the rate of beta-oxidation in heart can be regulated by the intramitochondrial ratio of [acetyl-CoA]/[CoASH] which reflects the energy demand of the tissue. The thiolytic cleavage catalyzed by 3-ketoacyl-CoA thiolase may be the site at which beta-oxidation is controlled by the [acetyl-CoA]/[CoASH] ratio.     

Elimination of CoASH interference from acetyl-CoA cycling assay by maleic anhydride

A method for the removal of CoASH from tissue extracts by maleic anhydride is described. It eliminates CoASH interference in the acetyl-CoA cycling assay using phosphotransacetylase and citrate synthase. Maleyl-CoA thioether does not hydrolyze under the conditions of the assay and allows a reduction in the number of blank samples during acetyl-CoA determination. The levels of acetyl-CoA in whole rat brain, isolated synaptosomes, and mitochondria were found to be 61, 8.6, and 31.3 pmol/mg of protein, respectively.     

Radioisotopic assay of picomolar amounts of coenzyme A

A two-step method of determining reduced coenzyme A (CoASH) concentrations in tissue or cell extracts is described. In the first step, CoASH is reacted with acetylphosphate in a reaction catalyzed by phosphotransacetylase to yield acetyl-CoA. Acetyl-CoA is then condensed with [14C]oxaloacetate by citrate synthase to give [14C]citrate. This method allows the measurement of 10-200 pmol of CoASH. By omitting the phosphotransacetylase step, measurement of the same amount of acetyl-CoA is possible.     

A sensitive radioisotopic assay of pyruvate dehydrogenase complex in human muscle tissue.

A radioactive assay for the determination of pyruvate dehydrogenase complex activity in muscle tissue has been developed. The assay measures the rate of acetyl-CoA formation from pyruvate in a reaction mixture containing NAD+ and CoASH. The acetyl-CoA is determined as [14C]citrate after condensation with [14C]-oxaloacetate by citrate synthase. The method is specific and sensitive to the picomole range of acetyl-CoA formed. In eleven normal subjects, the active form of pyruvate dehydrogenase (PDCa) in resting human skeletal muscle samples obtained using the needle biopsy technique was 0.44 +/- 0.16 (SD) mumol acetyl-CoA.min-1.g-1 wet wt. Total pyruvate dehydrogenase complex (PDCt) activity was determined after activation by pretreating the muscle homogenate with Ca2+, Mg2+, dichloroacetate, glucose, and hexokinase. The mean value for PDCt was 1.69 +/- 0.32 mumol acetyl-CoA.min-1.g-1 wet wt, n = 11. The precision of the method was determined by analyzing 4-5 samples of the same muscle piece. The coefficient of variation for PDCa was 8% and for PDCt 5%.     

Studies on the effects of coenzyme A-SH: acetyl coenzyme A, nicotinamide adenine dinucleotide: reduced nicotinamide adenine dinucleotide, and adenosine diphosphate: adenosine triphosphate ratios on the interconversion of active and inactive pyruvate dehydrogenase in isolated rat heart mitochondria.

The content of coenzyme A-SH (CoASH) and acetyl-CoA of suspensions of rat heart mitochondria was stabilized by the addition of DL-carnitine and acetyl-DL-carnitine, in the presence of the respiratory inhibitor rotenone. The mitochondrial content of NAD+ and NADH was similarly stabilized by the addition of acetoacetate and DL-3-hydroxybutyrate, and the content of ADP and ATP was imposed by the addition of these nucleotides to the mitochondrial suspension, in the presence of uncoupling agent and oligomycin, to inhibit ATPase. Under these conditions, mitochondrial CoASH/acetyl-CoA, NAD+/ NADH, and ADP/ATP ratios could be varied independently, and the effect on the interconversion of active and inactive pyruvate dehydrogenase could be studied. Decreases in both CoASH/acetyl-CoA and NAD+/NADH ratios were shown to be inhibitory to the steady state activity of pyruvate dehydrogenase, and this effect is described at three different ADP/ATP ratios and different concentrations of added MgCl2. A new steady state level of activity was achieved within 10 min of a change in either CoASH/acetyl-CoA or NAD+/NADH ratio; the rate of inactivation was much higher than the rate of reactivation under these conditions. Effects of CoASH/acetyl-CoA and NAD+/NADH may be additive but are still quantitatively lesser than the changes in activity of pyruvate dehydrogenase induced by changes in ADP/ATP ratio. The variation in activity of pyruvate dehydrogenase with ADP/ATP ratio is described in the absence of changes in the other two ratios, conditions which were not met in earlier studies which employed the oxidation of different substrates to generate changes in all three ratios.     

Regulation of pyruvate dehydrogenase by fatty acid in isolated rat liver mitochondria.

The mechanism by which fatty acid addition leads to the inactivation of pyruvate dehydrogenase in intact rat liver mitochondria was investigated. In all cases the fatty acid octanoate was added to mitochondria oxidizing succinate. Addition of fatty acid caused an inactivation of pyruvate dehydrogenase in mitochondria incubated under State 3 conditions (glucose plus hexokinase), in uncoupled, oligomycin-treated mitochondria, and in rotenone-menadione-treated mitochondria, but not in uncoupled mitochondria or in mitochondria incubated under State 4 conditions. A number of metabolic conditions were found in which pyruvate dehydrogenase was inactivated concomitant with an elevation in the ATP/ADP ratio. This is consistent with the inverse relationship between the ATP/ADP ratio and the pyruvate dehydrogenase activity proposed by various laboratories. However, in several other metabolic conditions pyruvate dehydrogenase was inactivated while the ATP/ADP ratio either was unchanged or even decreased. This observation implies that there are likely other regulatory factors involved in the fatty acid-mediated inactivation of pyruvate dehydrogenase. Incubation conditions in State 3 were found in which the ATP/ADP and the acetyl-CoA/CoASH ratios remained constant and the pyruvate dehydrogenase activity was correlated inversely with the NADH/NAD+ ratio. Other State 3 conditions were found in which the ATP/ADP and the NADH/NAD+ ratios remained constant while the pyruvate dehydrogenase activity was correlated inversely with the acetyl-CoA/CoASH ratio. Further evidence supporting these experiments with intact mitochondria was the observation that the pyruvate dehydrogenase kinase activity of a mitochondrial extract was stimulated strongly by acetyl-CoA and was inhibited by NAD+ and CoASH. In contrast to acetyl-CoA, octanoyl-CoA inhibited the kinase activity. These results indicate that the inactivation of pyruvate dehydrogenase by fatty acid in isolated rat liver mitochondria may be mediated through effects of the NADH/NAD+ ratio and the acetyl-CoA/CoASH ratio on the interconversion of the active and inactive forms of the enzyme complex catalyzed by pyruvate dehydrogenase kinase and pyruvate dehydrogenase phosphatase.     

Kinetics and properties of β-ketothiolase from Clostridium pasteurianum

1. Beta-Ketothiolase of Clostridium pasteurianum was purified 130-fold by ammonium sulphate fractionation and by column chromatography using DEAE-Sephadex A-50 and hydroxylapatite. Subjected to gel electrophoresis beta-ketothiolase revealed two distinct bands; by isoelectric focusing two enzymes with isoelectric points at pH 4.5 and 7.6 were separated. As established by sucrose density gradient centrifugation the molecular weight of both enzymes was found to be 158000. 2. The condensation reaction was measured by a coupled optical test using beta-hydroxybutyryl-CoA dehydrogenase as auxiliary enzyme and either acetyl-CoA or free coenzyme A plus acetyl-phosphate and phosphotransacetylase (regenerating system) or acetyl-CoA plus regenerating system as substrates. Beta-Ketothiolase from C. pasteurianum used only 20% of the chemically synthesized acetyl-CoA; the enzyme from Alcaligenes eutrophus H 16 used 25%. When the regenerating system was added the condensation reaction continued. The enzyme from C. pasteurianum was inactivated by free coenzyme A, while the enzyme from A. eutrophus was inhibited. When acetyl-CoA was added as the substrate the initial velocity determination was impeded by the lack of linearity. With acetyl-CoA as the substrate the Km-value was found to be 2.5 mM acetyl-CoA. If free CoASH (or acetyl-CoA) plus regenerating system was added the Km was 0.44 mM (0.42 mM) acetyl-CoA. 3. The beta-ketothiolase activity was measured in the direction of acetoacetyl-CoA cleavage by an optical assay following the decrease of the enol and chelate form of acetoacetyl-CoA by absorption measurement at 305 nm. The activity was maximal at 24 nM MgCl2. The apparent Km values for acetoacetyl-CoA were 0.133 mM and 0.105 mM with 0.065 and 0.016 mM CoASH, respectively. The Km-values as calculated for only the keto form of acetoacetyl-CoA were 0.0471 and 0.0372 mM, respectively. The cleavage reaction was inhibited by high acetoacetyl-CoA concentrations; the inihibition was partially relieved by CoASH. In the range of low concentrations of acetoacetyl-CoA only a slight inhibition by CoASH was observed. The Km for CoASH was found to be 0.0288 and 0.0189 mM with 0.09 and 0.045 mM acetoacetyl-CoA, respectively. High concentrations of CoASH exerted an inhibitory effect on the cleavage reaction. With respect to enzyme kinetics and sensitivity to inhibitors and metabolites the beta-ketothiolases of C. pasteurianum and A. eutrophus were rather similar.     

The NADP-dependent malic enzyme MaeB is a central metabolic hub controlled by the acetyl-CoA to CoASH ratio

Malic enzymes participate in key metabolic processes, the MaeB-like malic enzymes carry a catalytic inactive phosphotransacetylase domain whose function remains elusive. Here we show that acetyl-CoA directly binds and inhibits MaeB-like enzymes with a saturable profile under physiological relevant acetyl-CoA concentrations. A MaeB-like enzyme from the nitrogen-fixing bacterium Azospirillum brasilense, namely AbMaeB1, binds both acetyl-CoA and unesterified CoASH in a way that inhibition of AbMaeB1 by acetyl-CoA is relieved by increasing CoASH concentrations. Hence, AbMaeB1 senses the acetyl-CoA/CoASH ratio. We revisited E. coli MaeB regulation to determine the inhibitory constant for acetyl-CoA. Our data support that the phosphotransacetylase domain of MaeB-like enzymes senses acetyl-CoA to dictate the fate of carbon distribution at the phosphoenol-pyruvate / pyruvate / oxaloacetate metabolic node.     

Regulation of thiolases from pig heart. Control of fatty acid oxidation in heart

The effects of various mitochondrial coenzymes and metabolities on the activities of 3-oxoacyl-CoA thiolase (EC 2.3.1.16) and acetoacetyl-CoA thiolase (EC 2.3.1.9) from pig heart were investigated with the aim of elucidating the possible regulation of these two enzymes. Of the compounds tested, acetyl-CoA was the most effective inhibitor of both thiolases. However, 3-oxoacyl-CoA thiolase was more severly inhibited by acetyl-CoA than was acetoacetyl-CoA thiolase. 3-Oxoacyl-CoA thiolase was also significantly inhibited by decanoyl-CoA while acetoacetyl-CoA thiolase was inhibited by 3-hydroxybutyryl-CoA as strongly as it was by acetyl-CoA. All other compounds either did not affect the thiolase activities or only at unphysiologically high concentrations. The inhibition of acetoacetyl-CoA thiolase by acetyl-CoA was linear and apparently noncompetitive with respect to CoASH (Ki = 125 microM) whereas that of 3-oxoacyl-CoA thiolase was nonlinear. However at low concentrations of acetyl-CoA the inhibition of 3-oxoacyl-CoA thiolase was linear competitive with respect to CoASH (Ki = 3.9 microM). It is concluded that 3-oxoacyl-CoA thiolase, but not acetoacetyl-CoA thiolase, will be completely inhibited by acetyl-CoA at concentrations of CoASH and acetyl-CoA which are assumed to exist intramitochondrially at state-4 respiration. It is suggested that fatty acid oxidation in heart muscle at sufficiently high concentrations of plasma free fatty acids is controlled via the regulation of 3-oxoacyl-CoA thiolase by the acetyl-CoA/CoASH ratio which is determined by the rate of the citric acid cycle and consequently by the energy demand of the tissue.     

Regulation of ketogenesis. Mitochondrial acetyl-CoA acetyltransferase from rat liver: initial-rate kinetics in the presence of the product CoASH reveal intermediary plateau regions

The analysis of the initial-rate kinetics of the liver mitochondrial acetyl-CoA acetyltransferase (acetoacetyl-CoA thiolase) in the direction of acetoacetyl-CoA synthesis under product inhibition was performed. 1. Acetyl-CoA acetyltransferase shows a hyperbolic response of reaction velocity to changes in acetyl-CoA concentrations with an apparent Km of 0.237 +/- 0.001 mM. 2. CoASH is a (non-competitive) product inhibitor with a Kis of 22.6 microM and shifts the apparent Km for acetyl-CoA to the physiological concentration of this substrate in mitochondria (S0.5 = 1.12 mM in the presence of 121 microM CoASH). 3. CoASH causes a transformation of the Michaelis-Menten kinetics into initial-rate kinetics with four intermediary plateau regions. 4. The product analogue desulpho-CoA triggers a negative cooperativity as to the dependence of the reaction velocity on the acetyl-CoA concentration. These product effects drastically desensitize the acetyl-CoA acetyltransferase in its reaction velocity response to the acetyl-CoA concentrations and simultaneously extend the substrate dependence range. Thus a control of acetoacetyl-CoA synthesis by the substrate is established over the physiological acetyl-CoA concentration range. We suggest that this control mechanism is the key in establishing the rates of ketogenesis.     

ENZYMATIC FLUOROMETRIC DETERMINATION OF ACETYLCHOLINE IN BIOLOGICAL EXTRACTS1

Abstract— Acetylcholine was determined fluorometrically by the following enzymic reactions: (1) ACh = Acetate + Choline (2) Acetate + ATP + CoASH = Acetyl-SCoA + AMP + PPi (3) Malate + NAD⁺= Oxalacetate + NADH (4) Oxalacetate + Acetyl-SCoA = CoASH + Citrate The fluorescence produced by NADH was stoichiometric with the ACh present and the citrate formed. The complete system contained acetylcholinesterase (EC 3.1.1.7), acetyl-CoA synthetase (EC 6.2.1.1), malate dehydrogenase (EC 1.1.1.37), and citrate condensing enzyme (EC 4.1.3.7). The acetyl-CoA synthetase was rate limiting in the system. Authentic samples of ACh (10^?8 to 10^?9mol) were measured with ±5 per cent reproducibility; this corresponds to the content of an 80 mg (fresh weight) sample of brain. Tissue levels of ACh in this concentration range, within normal biological variation, were determined with ±15 per cent reproducibility. The method can also be employed to measure acetate and acetyl-SCoA with the same degrees of sensitivity and reproducibility. The method can be used to measure ‘total’ and ‘bound’ ACh and thus to estimate ‘free’ ACh, by varying the extraction procedure. Values obtained in the manner described agree with those previously reported in the literature. Troublesome fluorescence in brain extracts was effectively removed with acid-washed Florisil.     

Changes in the size and composition of intracellular pools of nonesterified coenzyme A and coenzyme A thioesters in aerobic and facultatively anaerobic bacteria.

Intracellular levels of three coenzyme A (CoA) molecular species, i.e., nonesterified CoA (CoASH), acetyl-CoA, and malonyl-CoA, in a variety of aerobic and facultatively anaerobic bacteria were analyzed by the acyl-CoA cycling method developed by us. It was demonstrated that there was an intrinsic difference between aerobes and facultative anaerobes in the changes in the size and composition of CoA pools. The CoA pools in the aerobic bacteria hardly changed and were significantly smaller than those of the facultatively anaerobic bacteria. On the other hand, in the facultatively anaerobic bacteria, the size and composition of the CoA pool drastically changed within minutes in response to the carbon and energy source provided. Acetyl-CoA was the major component of the CoA pool in the facultative anaerobes grown on sufficient glucose, although CoASH was dominant in the aerobes. Therefore, the acetyl-CoA/CoASH ratios in facultatively anaerobic bacteria were 10 times higher than those in aerobic bacteria. In Escherichia coli K-12 cells, the addition of reagents to inhibit the respiratory system led to a rapid decrease in the amount of acetyl-CoA with a concomitant increase in the amount of CoASH, whereas the addition of cerulenin, a specific inhibitor of fatty acid synthase, triggered the intracellular accumulation of malonyl-CoA. The acylation and deacylation of the three CoA molecular species coordinated with the energy-yielding systems and the restriction of the fatty acid-synthesizing system of cells. These data suggest that neither the accumulation of acetyl-CoA nor that of malonyl-CoA exerts negative feedback on pyruvate dehydrogenase and acetyl-CoA carboxylase, respectively.     

A shared binding site for NAD+ and coenzyme A in an acetaldehyde dehydrogenase involved in bacterial degradation of aromatic compounds

The meta-cleavage pathway for catechol is a central pathway for the bacterial dissimilation of a wide variety of aromatic compounds, including phenols, methylphenols, naphthalenes, and biphenyls. The last enzyme of the pathway is a bifunctional aldolase/dehydrogenase that converts 4-hydroxy-2-ketovalerate to pyruvate and acetyl-CoA via acetaldehyde. The structure of the NAD (+)/CoASH-dependent aldehyde dehydrogenase subunit is similar to that of glyceraldehyde-3-phosphate dehydrogenase, with a Rossmann fold-based NAD (+) binding site observed in the NAD (+)-enzyme complex [Manjasetty, B. A., et al. (2003) Proc. Natl. Acad. Sci. U.S.A. 100, 6992-6997]. However, the location of the CoASH binding site was not determined. In this study, hydrogen-deuterium exchange experiments, coupled with peptic digest and mass spectrometry, were used to examine cofactor binding. The pattern of hydrogen-deuterium exchange in the presence of CoASH was almost identical to that observed with NAD (+), consistent with the two cofactors sharing a binding site. This is further supported by the observations that either CoASH or NAD (+) is able to elute the enzyme from an NAD (+) affinity column and that preincubation of the enzyme with NAD (+) protects against inactivation by CoASH. Consistent with these data, models of the CoASH complex generated using AUTODOCK showed that the docked conformation of CoASH can fully occupy the cavity containing the enzyme active site, superimposing with the NAD (+) cofactor observed in the X-ray crystal structure. Although CoASH binding Rossmann folds have been described previously, this is the first reported example of a Rossmann fold that can alternately bind CoASH or NAD (+) cofactors required for enzymatic catalysis.     

Enzymatic determination of choline acetyltransferase by coenzyme A cycling and its application to analysis of single mammalian neurons

Abstract: An enzymatic assay for choline acetyltrans-ferase was developed by measuring acetyl-coenzyme A (acetyl-CoA) formed from CoASH and acetylcholine (ACh). This method is extremely sensitive and may be applied to the analysis of microgram to nanogram crude samples. The method is, however, not useful when choline acetyltransferase is present in very low concentrations. The basis of this method is to amplify a small amount of synthesized acetyl-CoA in the assay mixture by using an enzymatic amplification reaction, CoA cycling. This amplification mechanism made it possible to perform microassays (13 nl-2.2 μl of assay volume) of freeze-dried sections prepared from cerebral cortex, striatum, and hippocampus of mice and single cell bodies isolated from freeze-dried sections of rabbit spinal cords. These samples were weighed and added directly to the reaction mixture. The activities of the above cerebral regions, assayed with 1,500–2,000-fold amplification, corresponded well to the results previously reported by other workers. The average activity of single anterior horn cells, determined with 64,000–420,000-fold amplification, was 40-fold higher than that of rabbit cerebral cortex, and the specific activities on a dry weight basis were widely distributed among individual neurons. No activity was detected in the noncholinergic dorsal root ganglion cells or in cerebellar cortex.     

Short-chain acyl-CoA-dependent production of oxalate from oxaloacetate by Burkholderia glumae, a plant pathogen which causes grain rot and seedling rot of rice via the oxalate production.

In Burkholderia glumae (formerly named Pseudomonas glumae), isolated as the causal agent of grain rot and seedling rot of rice, oxalate was produced from oxaloacetate in the presence of short-chain acyl-CoA such as acetyl-CoA and propionyl-CoA. Upon purification, the enzyme responsible was separated into two fractions (tentatively named fractions II and III), both of which were required for the acyl-CoA-dependent production of oxalate. In conjugation with the oxalate production from oxaloacetate catalyzed by fractions II and III, acetyl-CoA used as the acyl-CoA substrate was consumed and equivalent amounts of CoASH and acetoacetate were formed. The isotope incorporation pattern indicated that the two carbon atoms of oxalate are both derived from oxaloacetate, and among the four carbon atoms of acetoacetate two are from oxaloacetate and two from acetyl-CoA. When the reaction was carried out with fraction II alone, a decrease in acetyl-CoA and an equivalent level of net utilization of oxaloacetate were observed without appreciable formation of CoASH, acetoacetate or oxalate. It appears that in the oxalate production from oxaloacetate and acetyl-CoA, fraction II catalyzes condensation of the two substrates to form an intermediate which is split into oxalate and acetoacetate by fraction III being accompanied by the release of CoASH.     

Regulation of pantothenate kinase by coenzyme A and its thioesters

Pantothenate kinase catalyzes the rate-controlling step in the coenzyme A (CoA) biosynthetic pathway, and its activity is modulated by the size of the CoA pool. The effect of nonesterified CoA (CoASH) and CoA thioesters on the activity of pantothenate kinase was examined to determine which component of the CoA pool is the most effective regulator of the enzyme from Escherichia coli. CoASH was five times more potent than acetyl-CoA or other CoA thioesters as an inhibitor of pantothenate kinase activity in vitro. Inhibition by CoA thioesters was not due to their hydrolysis to CoASH. CoASH inhibition was competitive with respect to ATP, thus providing a mechanism to coordinate CoA production with the energy state of the cell. There were considerable differences in the size and composition of the CoA pool in cells grown on different carbon sources, and a carbon source shift experiment was used to test the inhibitory effect of the different CoA species in vivo. A shift from glucose to acetate as the carbon source resulted in an increase in the CoASH:acetyl-CoA ratio from 0.7 to 4.3. The alteration in the CoA pool composition was associated with the selective inhibition of pantothenate phosphorylation, consistent with CoASH being a more potent regulator of pantothenate kinase activity in vivo. These results demonstrate that CoA biosynthesis is regulated through feedback inhibition of pantothenate kinase primarily by the concentration of CoASH and secondarily by the size of the CoA thioester pool.     

Modulation of rat-liver mitochondrial acetyl-CoA acetyltransferase activity by a reversible chemical modification with coenzyme A

The mitochondrial acetyl-CoA acetyltransferase (acetoacetyl-CoA thiolase, EC 2.3.1.9) is involved in ketone body biosynthesis. In its unmodified state, referred to as transferase B in former publications (Huth, W. (1981) Eur. J. Biochem. 120, 557-562), the enzyme is characterized by the highest specific activity of 21.65 mumol/min per mg protein (direction of acetoacetyl-CoA synthesis); several forms of the enzyme with lower specific activities result from chemical modification by an apparent covalent binding of CoASH. The chemical modification results in an inactivation of the enzyme: a 2 h incubation with 0.2 mM CoASH at pH 8.1 at 30 degrees C inactivates up to 95%. Both processes, the CoASH-binding and the resulting inactivation, can be simultaneously reversed by treatment with glutathione. The specificity of inactivation is limited to CoASH and the intact sulfhydryl group is a prerequisite for this process. The enzyme exhibits a limited number (n = 3.2) of high-affinity (Ka = 26.7 microM) specific binding sites for CoASH. The inactivation-reactivation cycle of acetyl-CoA acetyltransferase by CoASH and glutathione may involve a protein disulfide-thiol exchange and represents a mode of control in modulating the amount of active enzyme.     

On the mechanism of the chemical modification of the mitochondrial acetyl-CoA acetyltransferase by coenzyme A

The liver mitochondrial acetyl-CoA acetyltransferase (acetyl-CoA:acetyl-CoA C-acetyltransferase, EC 2.3.1.9), is involved in ketone body synthesis. The enzyme can be chemically modified and inactivated by CoASH and also by CoASH-disulfides provided glutathione is present. The unmodified enzyme shows in its denatured state 7.95 +/- 0.44 sulfhydryl groups per enzyme and in its native state 3.92 +/- 0.34 sulfhydryl groups which react with Ellmann's reagent. The modified enzyme reveals in its native state also 4.07 +/- 0.25 sulfhydryl groups per enzyme, but in its denatured state 9.10 +/- 0.51 sulfhydryl groups could be detected. Approximately four sulfhydryl groups per enzyme, unmodified or modified, can be alkylated by iodoacetamide. These results prove for each subunit the existence of two sulfhydryl groups and suggest the existence of two disulfide bridges. The CoASH modification, which should proceed at one of these disulfide groups, prevents subsequent acetylation of the enzyme and is drastically reduced in the iodoacetamide-alkylated enzyme. In the demodification of the modified enzyme, the CoASH is set free as a mixed disulfide with glutathione.     

Functional characterization and localization of acetyl-CoA hydrolase,Ach1p,in Saccharomyces cerevisiae

Acetyl-CoA hydrolase (Ach1p), catalyzing the hydrolysis of acetyl-CoA, is presumably involved in regulating intracellular acetyl-CoA or CoASH pools; however, its intracellular functions and distribution remain to be established. Using site-directed mutagenesis analysis, we demonstrated that the enzymatic activity of Ach1p is dependent upon its putative acetyl-CoA binding sites. The ach1 mutant causes a growth defect in acetate but not in other non-fermentable carbon sources, suggesting that Ach1p is not involved in mitochondrial biogenesis. Overexpression of Ach1p, but not constructs containing acetyl-CoA binding site mutations, in ach1-1 complemented the defect of acetate utilization. By subcellular fractionation, most of the Ach1p in yeast was distributed with mitochondria and little Ach1p in the cytoplasm. By immunofluorescence microscopy, we show that Ach1p and acetyl-CoA binding site-mutated constructs, but not its N-terminal deleted construct, are localized in mitochondria. Moreover, the onset of pseudohyphal development in homozygote ach1-1 diploids was abolished. We infer that Ach1p may be involved in a novel acetyl-CoA biogenesis and/or acetate utilization in mitochondria and thereby indirectly affect pseudohyphal development in yeast.