Hydrolysis Of acetyl-CoA Research Articles

Measurement of the rates of acetyl-CoA hydrolysis and synthesis from acetate in rat hepatocytes and the role of these fluxes in substrate cycling

1. Acetyl-CoA hydrolysis, acetyl-CoA synthesis from acetate and several related fluxes were measured in rat hepatocytes. 2. In contrast with acetyl-CoA hydrolysis, most of the acetyl-CoA synthesis from acetate occurred in the mitochondria. 3. Acetyl-CoA hydrolysis was not significantly affected by 24 h starvation or (-)-hydroxycitrate. 4. In the cytoplasm there was a net flux of acetyl-CoA to acetate, and substrate cycling between acetate and acetyl-CoA in this compartment was very low, accounting for less than 0.1% of the total heat production by the animal. 5. A larger cycle, involving mitochondrial and cytoplasmic acetate and acetyl-CoA, may operate in fed animals, but would account for only approx 1% of total heat production. 6. It is proposed that the opposing fluxes of mitochondrial acetate utilization and cytoplasmic net acetate production may provide sensitivity, feedback and buffering, even when these fluxes are not linked to form a conventional substrate cycle.

A glucose-repressible gene encodes acetyl-CoA hydrolase from Saccharomyces cerevisiae.

Acetyl-CoA hydrolase, catalyzing the hydrolysis of acetyl-CoA, is presumably involved in regulating the intracellular acetyl-CoA pool. Recently, a yeast acetyl-CoA hydrolase was purified to homogeneity from Saccharomyces cerevisiae and partially characterized (Lee, F.-J. S., Lin, L.-W., and Smith, J. A. (1989) Eur. J. Biochem. 184, 21-28). In order to study the biological function and regulation of the acetyl-CoA hydrolase, we cloned and sequenced the full length cDNA encoding yeast acetyl-CoA hydrolase. RNA blot analysis indicates that acetyl-CoA hydrolase is encoded by a 2.5-kilobase mRNA. DNA blot analyses of genomic and chromosomal DNA reveal that the gene (so-called ACH1, acetyl-CoA hydrolase) is present as a single copy located on chromosome II. Acetyl-CoA hydrolase is established to be a mannose-containing glycoprotein, which binds concanavalin A. By measuring the levels of ACH1 mRNA and acetyl-CoA hydrolase activity in different growth phases and by examining the effects of various carbon sources, we have demonstrated that ACH1 expression is repressed by glucose.

Purification and characterization of an acetyl-CoA hydrolase from Saccharomyces cerevisiae

Acetyl-CoA hydrolase, which hydrolyzes acetyl-CoA to acetate and CoASH, was isolated from Saccharomyces cerevisiae and demonstrated by protein sequence analysis to be NH2-terminally blocked. The enzyme was purified 1080-fold to apparent homogeneity by successive purification steps using DEAE-Sepharose, gel filtration and hydroxylapatite. The molecular mass of the native yeast acetyl-CoA hydrolase was estimated to be 64 +/- 5 kDa by gel-filtration chromatography. SDS/PAGE analysis revealed that the denatured molecular mass was 65 +/- 2 kDa and together with that for the native enzyme indicates that yeast acetyl-CoA hydrolase was monomeric. The enzyme had a pH optimum near 8.0 and its pI was approximately 5.8. Several acyl-CoA derivatives of varying chain length were tested as substrates for yeast acetyl-CoA hydrolase. Although acetyl-CoA hydrolase was relatively specific for acetyl-CoA, longer acyl-chain CoAs were also hydrolyzed and were capable of functioning as inhibitors during the hydrolysis of acetyl-CoA. Among a series of divalent cations, Zn2+ was demonstrated to be the most potent inhibitor. The enzyme was inactivated by chemical modification with diethyl pyrocarbonate, a histidine-modifying reagent.

A method for increasing the sensitivity of chloramphenicol acetyltransferase assays in extracts of transfected cultured cells

Transfection of several cell lines (HeLa, COS, PC-12, CA-77, and H4IIE C3) with pRSV-CAT by a variety of methods yielded rather low chloramphenicol acetyltransferase (CAT) activity in cell extracts. Extracts of these cells were found to interfere with the assay of added CAT. The extracts were capable of deacetylating acetylchloramphenicol and of accelerating the rate of hydrolysis of the acetyl-CoA present in the assay. Heating the cell extract to 60°C for 10 min completely prevented the interference and slowed the hydrolysis of acetyl-CoA. Substantially higher CAT activities were observed when the extract was heat treated in the presence of EDTA prior to enzyme assay for most cell lines tested. This simple reliable method makes possible the accurate assessment of CAT activities in different cell lines. These observations are particularly pertinent to investigators studying tissue-specific gene expression.

Measurement of free and esterified carnitine in tissue extracts by high-performance liquid chromatography

A high-performance liquid chromatographic (HPLC) method for the determination of l-carnitine in clamped and frozen rat livers is described. L-carnitine + acetyl-CoA ⇆ acetyk-camitine + CoASH Using the above enzymatic reaction, release of CoASH is stoichiometric with the l-carnitine added. The present method has made possible the determination of carnitine in liver tissues, which is difficult by the conventional enzymatic spectrophotometric method using 5,5′-dithiobis(2-nitrobenzoic acid), owing to acetyl-CoA hydrolysis during prolonged incubations at pH 7.8.

Pyruvate dehydrogenase complex of Escherichia coli. Thiamin pyrophosphate and NADH-dependent hydrolysis of acetyl-CoA.

When the pyruvate dehydrogenase complex of Escherichia coli is reduced by NADH and alkylated by N-[14C]ethylmaleimide, 19-20 nmol of N-[14C]ethylmaleimide are bound per mg of complex. This is in accord with the presence of 10 nmol of functional lipoyl moieties per mg of complex as previously reported. Thus the lipoyl groups are all coupled via dihydrolipoyl dehydrogenase (E3) to reduction by NADH. As previously reported, the complex reductively acetylated by pyruvate and containing 10 nmol of acetyldihydrolipoyl groups per mg of complex produces about 5 nmol of NADH/mg of complex when challenged with CoA and NAD+ in a fast burst. Under anaerobic conditions a slow secondary process extending over 1 h produces another 5 nmol of NADH/mg of complex. The relationship between the two classes of acetyldihydrolipoyl groups is unknown but could reflect either intrinsic structural inequivalence of lipoyl groups (2/subunit of dihydrolipoyl transacetylase, E2). Alternatively, the acetyldihydrolipoyl groups may undergo reversible isomerization to structurally distinct forms. The purified complex catalyzes the cleavage of acetyl-CoA by two processes. The trace contaminant phosphotransacetylase catalyzes cleavage by phosphate to acetyl-P. The complex itself catalyzes hydrolysis of acetyl-CoA in a reaction that requires all three enzymes, NADH, thiamin pyrophosphate, and the lipoyl groups of E2. The hydrolytic pathway evidently involves overall reversal of the reaction, leading ultimately to the formation of acetyl-thiamin pyrophosphate, which undergoes hydrolysis to acetate.

Carnitine in Trypanosoma brucei brucei

Bloodstream and culture forms of Trypanosoma brucei brucei contain high concentrations of intracellular l-carnitine, determined enzymatically (1.46 ± 0.28 and 4.49 ± 0.34 mM, respectively), a large proportion of which is non-esterified. Uptake of carnitine by bloodstream forms has been shown to take place against a concentration gradient. The K m and V max for carnitine uptake, which was saturable, were 180 μM and 0.242 nmol · min −1 · (mg cell protein) −1. Uptake was inhibited by the sulphydryl reagents p-chloromercuribenzoic acid (PCMB), N-ethylmaleimide (NEM), but not by salicylhydroxamic acid (SHAM), iodoacetic acid (IAA) or fluoride. Over the temperature range 10° – 30°C carnitine uptake possessed a coefficient of 45.4 ± 7.2 kJ · mol −1; between 30 and 40°C this decreased to approximately 7.0 kJ · mol −1. Both bloodstream and culture forms of T. b. brucei have high levels of carnitine-stimulated acetyl-CoA hydrolysis, i.e. carnitine acetyltransferase (CAT) activity. In the absence and presence of 3 mM l-carnitine, these activities were 9.73 ± 3.63 and 117.4 ± 22.4, and 36.13 ± 9.43 and 524.4 ± 99.9 nmol · min −1 · ( mg cell protein) −1, respectively. CAT activity in bloodstream forms was half-maximally stimulated by an l-carnitine concentration of 0.79 mM. Two separate enzyme activities capable of hydrolysing acetyl-CoA have been distinguished based on their sensitivity to freezing and thawing. Acetyl-CoA hydrolysis in the absence of l-carnitine (thioesterase activity) showed an apparent K m for acetyl-CoA of 120 μM and a relatively low value for V max; this activity was very labile. Hydrolysis of acetyl-CoA in the presence of saturating concentrations of l-carnitine (20 mM; ≈ 25 K m) gave an apparent K m of 36 μM and a much higher value for V max; this activity was relatively stable on storage. CAT activity in bloodstream forms was shown to be competitively inhibited by ATP with a K i of 0.46 ± 0.03 mM. Total and free carnitine within the cell were measured for bloodstream and culture forms under various metabolic conditions. Acetyl group pressure decreased the proportion of free carnitine within the cell. It is suggested that the carnitine-CAT system in T. b. brucei has the physiological role of protecting the limited cellular CoA pool from metabolic ‘acetyl pressure’. Attempts to measure the cellular CoA levels in either bloodstream or culture forms were unsuccessful; however, an upper limit of 0.1 and 0.2 nmol CoA · (mg cell protein) −1, respectively can be put on the size of the cellular pool.

The relation of acyl transfer to the overall reaction of thiolase I from porcine heart.

Thiolase I (long chain 3-ketoacyl-CoA-specific) from porcine heart has been characterized kinetically. In the direction of acetoacetyl-CoA cleavage, a variety of thiols including CoASH show the same Vmax at saturating concentrations of acetoacetyl-CoA. At a constant overall velocity of acetoacetyl-CoA disappearance, one of the two acetyl groups from acetoacetyl-CoA will partition between CoASH and 2-mercaptoethanol at increasing 2-mercaptoethanol concentrations. These observations suggest rate-determining formation of an acetyl enzyme intermediate in the direction of acetoacetyl-CoA cleavage. In the direction of acetoacetyl-CoA formation from two molecules of acetyl-CoA, the Vmax of acetoacetyl-CoA formation is identical with the Vmax for an acetyl-CoA in equilibrium CoA isotope exchange reaction and the Vmax for an enzyme-catalyzed acetyl transfer reaction between acetyl-CoA and 2-mercaptoethanol. This suggests that in the direction of acetoacetyl-CoA synthesis, the acetyl transfer half-reaction is rate-limiting. The acetyl intermediate has been isolated and characterized. The equilibrium constant for acetyl enzyme formation from acetyl-CoA and free enzyme is 1 +/- 0.5 X 10(-2). The rate constant for spontaneous hydrolysis of the acetyl enzyme (2.6 X 10(-4) s-1) is a factor of 400 faster than the rate constant for acetyl-CoA hydrolysis under comparable conditions. The acetyl enzyme is thermodynamically and kinetically destabilized compared to acetyl-CoA.

Deacylation of acetyl-coenzyme A and acetylcarnitine by liver preparations.

The breakdown of acetylcarnitine catalysed by extracts of rat and sheep liver was completely abolished by Sephadex G-25 gel filtration, whereas the hydrolysis of acetyl-CoA was unaffected. Acetyl-CoA and CoA acted catalytically in restoring the ability of Sephadex-treated extracts to break down acetylcarnitine, which was therefore not due to an acetylcarnitine hydrolase but to the sequential action of carnitine acetyltransferase and acetyl-CoA hydrolase. Some 75% of the acetyl-CoA hydrolase activity of sheep liver was localized in the mitochondrial fraction. Two distinct acetyl-CoA hydrolases were partially purified from extracts of sheep liver mitochondria. Both enzymes hydrolysed other short-chain acyl-CoA compounds and succinyl-CoA (3-carboxypropionyl-CoA), but with one acetyl-CoA was the preferred substrate.

Alanine release by rat hemidiaphragm muscle in vitro.

than 3 m ~ , especially in T. cruzi, on the basis of the assumption that we were indeed measuring carnitine acetyltransferase activity in our preparations. Values of acetyl-CoA deacylase activity for various trypanosomatid flagellates are given in Table 1 together with the effect of adding 3 mM-L(-)-Carnitine where appropriate. Initial experiments have suggested that the deacylation of acetyl-CoA may be sensitive to the presence of added nucleoside diand tri-phosphates. In the absence of added L(-)carnitine GDP was stimulatory, whereas ADP and ATP were inhibitory. Typical results for 0 . 5 m ~ added nucleoside phosphate were for T. 6. brucei MIAG 103, GDP +loo% and ADP -32 %, and for T. b. brucei 427, GDP +75 %, ADP -31 % and ATP -49 %. In the presence of added L(-)-carnitine results appeared equivocal. Attempts to demonstrate substrate-level phosphorylation of GDP analogous to that observed in Trichomonus spp. containing hydrogenosomes (Muller, 1975) were unsuccessful. The significance of the stimulation by carnitine of acetyl-CoA hydrolysis may be related to either the site of infection, especially for T. cruzi, as suggested by Wood & Schiller (1975) or a mechanism for relieving ‘acetyl pressure’ (Costa & Snoswell, 1975) during the breakdown of large amounts of threonine.

3-Hydroxy-3-methylglutaryl coenzyme A synthase. Evidence for an acetyl-S-enzyme intermediate and identification of a cysteinyl sulfhydryl as the site of acetylation.

Homogeneous liver 3-hydroxy-3-methylglutaryl coenzyme A synthase, which catalyzes the condensation of acetyl-CoA with acetoacetyl-CoA to form 3-hydroxy-3-methylglutaryl-CoA, also carries out: (a) a rapid transacetylation from acetyl-CoA to 31-dephospho-CoA and (b) a slow hydrolysis of acetyl-CoA to acetate and CoA. Transacetylation and hydrolysis occur at 50 and 1 percent, respectively, the rate of the synthasecatalyzed condensation reaction. It appears that an acetyl-enzyme intermediate is involved in the transacetylase and hydrolase reactions of 3-hydroxy-3-methylglutaryl-CoA synthase, as well as in the over-all condensation process. Covalent binding to the enzyme of a [14C]acetyl group contributed by [1(-14)C]acetyl-CoA is indicated by migration of the [14C]acetyl group with the dissociated synthase upon electrophoresis in dodecyl sulfate-urea and by precipitation of [14C]acetyl-enzyme with trichloroacetic acid. At 0 degrees and a saturating level of acetyl-CoA, the synthase is rapidly (less than 20 s) acetylated yielding 0.6 acetyl group/enzyme dimer. Performic acid oxidation completely deacetylates the enzyme, suggesting the site of acetylation to be a cysteinyl sulfhydryl group. Proteolytic digestion of [14C]acetyl-S-enzyme under conditions favorable for intramolecular S to N acetyl group transfer quantitatively liberates a labeled derivative with a [14C]acetyl group stable to performic acid oxidation. The labeled oxidation product is identified as N-[14C]acetylcysteic acid, thus demonstrating a cysteinyl sulfhydryl group as the original site of acetylation. The ability of the acetylated enzyme, upon addition of acetoacetyl-CoA, to form 3-hydroxy-3-methylglutaryl-CoA indicates that the acetylated cysteine residue is at the catalytic site.

Equilibrium constants of the reactions of choline acetyltransferase, carnitine acetyltransferase, and acetylcholinesterase under physiological conditions.

The observed equilibrium constant (Kobs) for the reaction of choline acetyltransferase (EC 2.3.1.6) has been determined under physiological conditions. Using sigma and square brackets to indicate total concentrations of all ionic species present: (see article). The value of Kobs has been determined to be 12.3 plus or minus 0.6 at 38 degrees, pH 7.0 and ionic strength 0.25 M. The value at 25 degrees is not significantly different, and the constant has been found to be insensitive to variations in ionic strength (0.03 to 0.375 M), pH (6.5 TO 7.5) OR FREE [Mg-2+] (0 to 5 mM). The Kobs of this reaction reflects the difference between the observed standard free energy change (delta G-oobs) for the hydrolysis of acetylcholine and the delta G-oobs for the hydrolysis of acetyl-CoA. Since the delta G-oobs for the hydrolysis of acetyl-CoA has been previously determined to be minus 8.54 kcal/mol (minus 35.75 kJ/mol under the same physiological conditions, the delta G-oobs for the reaction of acetylcholinesterase (EC 3.1.1.7): (SEE ARTICLE). Can be calculated to be minus 6.99 kcal/mol (minus 29.26 kJ/mol) at pH ionic strength 0.25 M and 38 degrees, taking the standard state of liquid water to have unit activity ([H2O] equals 1). The pKa for acetic acid under the same conditions, has been determined to be 4.60 plus or minus 0.01, allowing the Kobs for the pH-independent reaction (see article). To be calculated to be 3.28 times 10-2 M. Choline and carnitine are chemical analogues. The Kobs for the corresponding reaction of carnitine acetyltransferase (EC 2.3.1.7). (SEE ARTICLE). Under the same physiological conditions of pH (7.0), ionic strength (0.25 M), and temperature (38 degrees) has been determined to be 1.73 plus or minus 0.05, making the delta G-oobs for the hydrolysis of acetylcholine only 1.21 kcal/mol (5.06 kJ) less negative than that for the hydrolysis of acetylcarnitine.

Production and utilization of acetate in mammals.

1. In an attempt to define the importance of acetate as a metabolic precursor, the activities of acetyl-CoA synthetase (EC 6.2.1.1) and acetyl-CoA hydrolase (Ec 3.1.2.1) were assayed in tissues from rats and sheep. In addition, the concentrations of acetate in blood and liver were measured, as well as the rates of acetate production by tissue slices and mitochondrial fractions of these tissues. 2. Acetyl-CoA synthetase occurs at high activities in heart and kidney cortex of both species as well as in rat liver and the sheep masseter muscle. The enzyme is mostly in the cytosol fraction of liver, whereas it is associated with the mitochondrial fraction in heart tissue. Both mitochondrial and cytosol activities have a K(m) for acetate of 0.3mm. Acetyl-CoA synthetase activity in liver was not altered by changes in diet, age or alloxan-diabetes. 3. Acetyl-CoA hydrolase is widely distributed in rat and sheep tissues, the highest activity being found in liver. Essentially all of the activity in liver and heart is localized in the mitochondrial fraction. Hepatic acetyl-CoA hydrolase activity is increased by starvation in rats and sheep and during the suckling period in young rats. 4. The concentrations of acetate in blood are decreased by starvation and increased by alloxan-diabetes in both species. The uptake of acetate by the sheep hind limb is proportional to the arterial concentration of acetate, except in alloxan-treated animals, where uptake is impaired. 5. Acetate is produced by liver and heart slices and also by heart mitochondrial fractions that are incubated with either pyruvate or palmitoyl-(-)-carnitine. Liver mitochondrial fractions do not form acetate from either substrate but instead convert acetate into acetoacetate. 6. We propose that acetate in the blood of rats or starved sheep is derived from the hydrolysis of acetyl-CoA. Release of acetate from tissues would occur under conditions when the function of the tricarboxylic acid cycle is restricted, so that the circulating acetate serves to redistribute oxidizable substrate throughout the body. This function is analogous to that served by ketone bodies.

La germination de la spore de clostridum tyrobutyricum: II. Démonstration de l'intervention de l'acétokinase et de la phosphotransacétylase par l'étude de leurs propriétés

Summary The properties of an acetokinase AK (ATP: acetate phosphotransferase, E.C. 2.7.2.1) and a phosphotransacetylase PTA (acetyl-CoA: orthophosphate acetyl transferase, E.C. 2.3.1.8) isolated from vegetative cells of C. tyrobutyricum have been studied in order to investigate if they are involved in spore germination. These enzymes are similar to those from other microrganisms especially. E. coli for AK and C. kluyveri for PTA. AK has a molecular weight of 68,000 daltons. It phosphorylates acetate and at a smaller rate propionate. The K m values are 100 mM for acetate, 135 mM for propionate, 3 mM for ATP, 5 mM for ADP and 3 mM for acetyl-P. The equilibrium constant of the AK reaction, which is markedly in favor of ATP and acetate, has been estimated to be 0.005 and the mechanism has been found to be «Ping-Pong. AK has an absolute requirement for Mg 2+ or Mn 2+ ; it is strongly inhibited by Mg + and p-CMB and activated by reducing agents. The phosphorylation of acetate by AK is inhibited competitively by the following compounds: fluoroacetate > butyrate ≥ thioglycolate > lactate > glycolate and propionate; formate, glycine, iodoacetate and acetamide have no effect. PTA has a molecular weight of 58,000 daltons. The reaction catalyzed by PTA is reversible but the rate of acetylation of CoA is much greater than the hydrolysis of acetyl-CoA. The K m values are 1.1 mM for acetyl-P and 0.03 mM for CoA. K + and particularly NH 4 + activates PTA, but Na + , Li + , Cs + and Ca 2+ inhibit it. Na + is a competitive inhibitor with respect to NH 4 + . Enzyme activity is also dependent on the anions, the relative order of effectiveness is: Cl − > CH 3 -COO − > SO 4 2− > PO 4 3− . NADH, ADP, ATP and pyruvate display no significant effect on PTA but a non-competitive inhibition with acetyl-P was observed with 3 and 2.3-PGA and a mixed inhibition with 2-PGA. The comparison of cell and spores AK and PTA on the basis of their pH optima and their interaction with substrates and inhibitors did not show significant differences. It is concluded that AK and PTA are involved in the initiation of spore germination in C. tyrobutyricum because of the complete agreement between several properties of these enzymes, especially the effect of inhibitors, and those of germination. An hypothesis on the role of acetyl-CoA is suggested.

Equilibrium Constants of the Reactions of Acetyl Coenzyme A Synthetase and the Hydrolysis of Adenosine Triphosphate to Adenosine Monophosphate and Inorganic Pyrophosphate

Abstract The observed standard free energy change (ΔG0obs) for the ATP-pyrophosphorylase reaction (EC 3.6.1.8) has been calculated for near physiological conditions of temperature, ionic strength, and free magnesium concentration. The observed equilibrium constant (Kobs) for the acetyl-CoA synthetase reaction (EC 6.2.1.1) has been determined at both 25 and 38°, pH 7.0, ionic strength 0.25, and varying free [Mg2+]. The Kobs of this reaction reflects the difference between the ΔG0obs for the hydrolysis of acetyl-CoA and the ΔG0obs for the hydrolysis of ATP to AMP and inorganic PPi. Using Σ and square brackets to indicate total concentrations of all the ionic species present: [see PDF for equation] The observed value of this constant varies with the free [Mg2+] being 10.7 (38°) and 15.1 (25°) at free [Mg2+] = 0; 9.17 (38°) and 12.9 (25°) at free [Mg2+] = 10-4 m, and 9.88 (38°) and 11.4 (25°) at free [Mg2+] = 10-3 m. The values of ΔH0obs for this reaction are also a function of the free [Mg2+], being 4.9 Cal per mole (20.5 kJ per mole) at free [Mg2+] = 0; 4.8 Cal per mole (20.0 kJ per mole) at free [Mg2+] = 10-4 m; and 3.6 Cal per mole (15.3 kJ per mole) at free [Mg2+] = 10-3 m. The ΔG0obs for the hydrolysis of acetyl-CoA is virtually unaffected by the free [Mg2+] and has been previously determined to be -8.54 Cal per mole (-35.75 kJ per mole) under the same conditions of temperature, pH, and ionic strength. Therefore, at 38°, pH 7.0, ionic strength 0.25, and taking the standard state of liquid water to have unit activity ([H2O] = 1), the ΔG0obs for the reaction [see PDF for equation] can be calculated at 38° and ionic strength of 0.25 to be -10.0 Cal per mole (-41.84 kJ per mole) at free [Mg2+] = 0; -9.91 Cal per mole (-41.46 kJ per mole) at free [Mg2+] = 10-4 m; and -9.96 Cal per mole (-41.67 kJ per mole) at free [Mg2+] = 10-3 m. The corresponding equilibrium constants for the hydrolysis of ATP to AMP and inorganic pyrophosphate under these conditions are 10.8 x 106 m ([Mg2+] = 0), 9.26 x 106 m (free [Mg2+] = 10-4 m), and 9.98 x 106 m (free [Mg2+] = 10-3 m).

Equilibrium Constants of the Malate Dehydrogenase, Citrate Synthase, Citrate Lyase, and Acetyl Coenzyme A Hydrolysis Reactions under Physiological Conditions

Abstract The observed equilibrium constants (Kobs) of the malate dehydrogenase (EC 1.1.1.37), citrate synthase (EC 4.1.3.7) and citrate lyase (EC 4.1.3.6) reactions have been determined under near physiological conditions (38°, pH 7.0, I = 0.25, free [Mg2+] = 10-3 m). From these values the observed standard free energy change (ΔG0obs) for the hydrolysis of acetyl-CoA has been determined. Under the above conditions, and taking the standard state of liquid water to have activity = unity ([H2O] = 1), the equilibrium constants of the three reactions at pH 7.0 may be written: Malate dehydrogenase Kobs = [Oxaloacetate][NADH]/[Malate][NAD+] = 2.86 ± 0.12 x 10-5 Citrate synthase Kobs = [Citrate][CoA]/[Oxaloacetate][Acetyl-CoA][H2O] = 2.24 ± 0.11 x 106 Citrate lyase Kobs = [Citrate]/[Oxaloacetate][Acetate] = 2.22 ± 0.16 m-1 where the concentration of each substrate represents the total of all the ionic species present, both acid forms and magnesium complexes. The values obtained for Kobs of the citrate synthase and citrate lyase reactions vary to the same extent with the changes in magnesium concentration. At free [Mg2+] = 0, Kobs for the citrate synthase reaction is 1.01 ± 0.05 x 106 and the Kobs for the citrate lyase reaction is 1.00 ± 0.07 m-1. In contrast, malate dehydrogenase is unaffected by the concentration of free Mg2+ up to 4 mm. From the constants of the citrate synthase and citrate lyase reactions, the Kobs for the hydrolysis of acetyl-CoA under near physiological conditions is calculated to be 1.01 x 106 m, corresponding to a free energy change of -8.54 Cal per mole (-35.75 kJ per mole) independent of the free magnesium concentration.

The Equilibrium Constants of the Adenosine Triphosphate Hydrolysis and the Adenosine Triphosphate-Citrate Lyase Reactions

Abstract The observed standard free energy change (ΔG0obs) for the hydrolysis of the terminal pyrophosphate bond of ATP has been experimentally determined under physiological conditions using an entirely new set of reactions. The observed equilibrium constant (Kobs) for the combined reactions of acetate kinase (EC 2.7.2.1) and phosphate acetyltransferase (EC 2.3.1.8) has been determined at 38°, pH 7.0, ionic strength 0.25, and varying free [Mg2+]. The Kobs of these combined reactions reflects the difference between ΔG0obs for the hydrolysis of acetyl-CoA and the ΔG0obs for the hydrolysis of ATP. Using Σ and square brackets to indicate total concentration, Kobs = [ΣADP][ΣPi]/[ΣATP] x [Acetyl-CoA]/[ΣAcetate][CoA] The observed value of this combined equilibrium constant varies with free [Mg2+], being 0.984 ± 0.009 when [Mg2+] = 0 and 0.218 ± 0.002 when free [Mg2+] = 10-3 m. The ΔG0obs for the hydrolysis of acetyl-CoA is virtually unaffected by the free [Mg2+] and has been previously determined to be -8.54 Cal per mole (-35.75 kJ per mole) under the same conditions of temperature, pH, and ionic strength. Therefore at pH 7.0, at ionic strength 0.25, at 38°, and taking the standard state of liquid water to have activity = unity ([H2O] = 1) the ΔG0obs for the reaction [see PDF for equation] can be calculated to be -8.53 Cal per mole (-35.69 kJ per mole) at [Mg2+] = 0 and -7.60 Cal per mole (-31.80 kJ per mole) at [Mg2+] = 10-3 m. The corresponding values of Kobs for the ATP hydrolysis reaction are 9.86 x 105 m ([Mg2+] = 0) and 2.19 x 105 m ([Mg2+] = 10-3 m). Equations have been developed for calculating from the experimental data the ΔG0obs of ATP hydrolysis at different free magnesium and hydrogen ion concentrations. The Kobs of ATP hydrolysis has been used in combination with the Kobs of the citrate synthase reaction (EC 4.1.3.7) to calculate the Kobs of the ATP-citrate lyase reaction (EC 4.1.3.8) Kobs = [ΣADP][Acetyl-CoA] [ΣPi] [ΣOxaloacetate]/[ΣATP] [CoA] [ΣCitrate] Under the same near physiological conditions of 38°, pH 7.0, and ionic strength 0.25, the value of Kobs for the ATP-citrate lyase reaction was found to be very sensitive to the free [Mg2+], being 0.975 m at [Mg2+] = 0 and 0.0985 m when free [Mg2+] = 10-3 m.

THE EQUILIBRIUM OF THE REACTION CATALYSED BY CITRATE OXALOACETATE-LYASE.

1. A method of preparation and purification of citrate oxaloacetate-lyase (EC 4.1.3.6) from Aerobacter aerogenes is described. 2. The equilibrium of this reaction has been determined at pH 8.4 and 25 degrees . It has been shown that K, i.e. [citrate(3-)]/[oxaloacetate(keto) (2-)][acetate (-)], is 3.08+/-0.72, but that K(app.), i.e. [total citrate]/[total oxaloacetate][total acetate], is markedly affected by the initial concentrations of the reactants and magnesium. 3. The free-energy change during the cleavage of citrate has been calculated and compared with data from other sources. 4. The free energy of hydrolysis of acetyl-CoA has been evaluated from the present data. 5. A detailed knowledge of the interactions of the reactants with metal ions has been shown to be important in the calculation of the equilibrium constant and related thermodynamic functions.