Purification, properties and substrate specificity of adenosine triphosphate sulphurylase from spinach leaf tissue

1. ATP sulphurylase was purified up to 1000-fold from spinach leaf tissue. Activity was measured by sulphate-dependent [(32)P]PP(i)-ATP exchange. The enzyme was separated from Mg(2+)-requiring alkaline pyrophosphatase (which interferes with the PP(i)-ATP-exchange assay) and from other PP(i)-ATP-exchange activities. No ADP sulphurylase activity was detected. 2. Sulphate was the only form of inorganic sulphur that catalysed PP(i)-ATP exchange; K(m) (sulphate) was 3.1mm, K(m) (ATP) was 0.35mm and the pH optimum was 7.5-9.0. The enzyme was insensitive to thiol-group reagents and required either Mg(2+) or Co(2+) for activity. 3. The enzyme catalysed [(32)P]PP(i)-dATP exchange; K(m) (dATP) was 0.84mm and V (dATP) was 30% of V (ATP). Competition between ATP and dATP was demonstrated. 4. Selenate catalysed [(32)P]PP(i)-ATP exchange and competed with sulphate; K(m) (selenate) was 1.0mm and V (selenate) was 30% of V (sulphate). No AMP was formed with selenate as substrate. Molybdate did not catalyse PP(i)-ATP exchange, but AMP was formed. 5. Synthesis of adenosine 5'-[(35)S]sulphatophosphate was demonstrated by coupling purified ATP sulphurylase and Mg(2+)-dependent alkaline pyrophosphatase (also prepared from spinach) with [(35)S]sulphate and ATP as substrates; adenosine 5'-sulphatophosphate was not synthesized in the absence of pyrophosphatase. Some parameters of the coupled system are reported.     

Microsomal palmitoyl-coenzyme A synthetase from rat liver. Partial and exchange reactions

The partial and exchange reactions of long-chain fatty acid activation were determined by using purified microsomal long-chain fatty acyl-CoA synthetase (EC 6.2.1.3). No significant ATP formation from palmitoyl-AMP and PP(i), nor palmitoyl-AMP-dependent CoA disappearance could be demonstrated. Similarly, no palmitate-dependent [(32)P(2)]PP(i)-ATP exchange was catalysed by the pure enzyme. The above partial and exchange reactions were, however, catalysed by the parent microsomal fraction at a rate similar to that of the overall reaction. The implications of these results are discussed.     

The enzymology of short-chain fatty acyl-coenzyme A synthetase from seeds of Pinus radiata. Kinetic studies and a proposed reaction mechanism

1. Short-chain fatty acyl-CoA synthetase from seeds of Pinus radiata was examined by acetate- and propionate-dependent PP(i)-ATP exchange. Reaction mixtures came to equilibrium almost instantly as judged by rates of exchange and analysis of an incubation mixture. 2. The activity of the enzyme was correlated with the concentration of MgP(2)O(7) (2-) but not with the concentration of Mg(2+), as judged by PP(i)-ATP exchange and fatty acyl AMP-dependent synthesis of ATP in the presence of PP(i). In PP(i)-ATP exchange assays, no clear relationship between activity and any single species of ATP was apparent. 3. High concentrations of fatty acid inhibited PP(i)-ATP exchange. PP(i)-dATP exchange was less than PP(i)-ATP exchange at low concentrations of fatty acid, but at higher concentrations PP(i)-dATP exchange exceeded PP(i)-ATP exchange. The rate of synthesis of fatty acyl-CoA in the presence of dATP was less than with ATP. 4. ATP and propionate inhibited the synthesis of ATP from propionyl-AMP and PP(i). The inhibition by ATP was competitive with respect to propionyl-AMP and non-competitive with respect to PP(i). The inhibition by propionate was non-competitive with respect to propionyl-AMP and PP(i). 5. AMP was a competitive inhibitor of propionyl-AMP-dependent synthesis of ATP and competitively inhibited propionate-dependent PP(i)-ATP exchange when ATP was the variable substrate. 6. It was concluded that the first partial reaction catalysed by the enzyme is ordered; ATP is the first substrate to react with the enzyme and PP(i) is probably the only product released.     

Comparative enzymology of the adenosine triphosphate sulphurylases from leaf tissue of selenium-accumulator and non-accumulator plants

1. ATP sulphurylases were partially purified (20-40-fold) from leaf tissue of Astragalus bisulcatus, Astragalus racemosus (selenium-accumulator species) and Astragalus hamosus and Astragalus sinicus (non-accumulator species). Activity was measured by sulphate-dependent PP(i)-ATP exchange. The enzymes were separated from pyrophosphatase and adenosine triphosphatase activities. The properties of the Astragalus ATP sulphurylases were similar to the spinach enzyme. 2. The ATP sulphurylases from both selenium-accumulator and non-accumulator species catalysed selenate-dependent PP(i)-ATP exchange; selenate competed with sulphate. The ratio of V(selenate)/V(sulphate) and K(m)(selenate)/K(m)(sulphate) was approximately the same for the enzyme from each species. 3. Sulphate-dependent PP(i)-ATP exchange was inhibited by ADP, chlorate and nitrate. The kinetics of the inhibition for each enzyme were consistent with an ordered reaction mechanism, in which ATP is the first substrate to react with the enzyme and PP(i) is the first product released. 4. Synthesis of adenosine 5'-[(35)S]sulphatophosphate from [(35)S]sulphate was demonstrated by coupling the Astragalus ATP sulphurylases with Mg(2+)-dependent pyrophosphatase; the reaction was inhibited by selenate. An analogous reaction using [(75)Se]selenate as substrate could not be demonstrated.     

The distribution of l-asparagine synthetase in the principal organs of several mammalian and avian species

1. Sulphate-dependent PP(i)-ATP exchange, catalysed by purified spinach leaf ATP sulphurylase, was correlated with the concentration of MgATP(2-) and MgP(2)O(7) (2-); ATP sulphurylase activity was not correlated with the concentration of free Mg(2+). 2. Sulphate-dependent PP(i)-ATP exchange was independent of PP(i) concentration, but dependent on the concentration of ATP and sulphate. The rate of sulphate-dependent PP(i)-ATP exchange was quantitatively defined by the rate equation applicable to the initial rate of a bireactant sequential mechanism under steady-state conditions. 3. Chlorate, nitrate and ADP inhibited the exchange reaction. The inhibition by chlorate and nitrate was uncompetitive with respect to ATP and competitive with respect to sulphate. The inhibition by ADP was competitive with respect to ATP and non-competitive with respect to sulphate. 4. ATP sulphurylase catalysed the synthesis of [(32)P]ATP from [(32)P]PP(i) and adenosine 5'-sulphatophosphate in the absence of sulphate; some properties of the reaction are described. Enzyme activity was dependent on the concentration of PP(i) and adenosine 5'-sulphatophosphate. 5. The synthesis of ATP from PP(i) and adenosine 5'-sulphatophosphate was inhibited by sulphate and ATP. The inhibition by sulphate was non-competitive with respect to PP(i) and adenosine 5'-sulphatophosphate; the inhibition by ATP was competitive with respect to adenosine 5'-sulphatophosphate and non-competitive with respect to PP(i). It was concluded that the reaction catalysed by spinach leaf ATP sulphurylase was ordered; expressing the order in the forward direction, MgATP(2-) was the first product to react with the enzyme and MgP(2)O(7) (2-) was the first product released. 6. The expected exchange reaction between sulphate and adenosine 5'-sulphatophosphate could not be demonstrated.     

Biochemical effects of the hypoglycaemic compound pent-4-enoic acid and related non-hypoglycaemic fatty acids. Effects of their coenzyme A esters on enzymes of fatty acid oxidation

1. Pent-4-enoyl-CoA and its metabolites penta-2,4-dienoyl-CoA and acryloyl-CoA, as well as n-pentanoyl-CoA, cyclopropanecarbonyl-CoA and cyclobutanecarbonyl-CoA, were examined as substrates or inhibitors of purified enzymes of beta-oxidation in an investigation to locate the site of inhibition of fatty acid oxidation by pent-4-enoate. 2. The reactions of various acyl-CoA derivatives with l-carnitine and of various acyl-l-carnitine derivatives with CoA, catalysed by carnitine acetyltransferase, were investigated and V(max.) and K(m) values were determined. Pent-4-enoyl-CoA and n-pentanoyl-CoA were good substrates, whereas cyclobutanecarbonyl-CoA, cyclopropanecarbonyl-CoA and acryloyl-CoA reacted more slowly. A very slow rate with penta-2,4-dienoyl-CoA was detected. Pent-4-enoyl-l-carnitine, n-pentanoyl-l-carnitine and cyclobutanecarbonyl-l-carnitine were good substrates and cyclopropanecarbonyl-l-carnitine reacted more slowly. 3. Pent-4-enoyl-CoA and n-pentanoyl-CoA were substrates for butyryl-CoA dehydrogenase and for octanoyl-CoA dehydrogenase, and both compounds were equally effective competitive inhibitors of these enzymes with butyryl-CoA or palmitoyl-CoA respectively as substrates. V(max.), K(m) and K(i) values were determined. 4. None of the acyl-CoA derivatives inhibited enoyl-CoA hydratase or 3-hydroxybutyryl-CoA dehydrogenase. Penta-2,4-dienoyl-CoA was a substrate for enoyl-CoA hydratase when the reaction was coupled to that catalysed by 3-hydroxybutyryl-CoA dehydrogenase. 5. In a reconstituted sequence with purified enzymes crotonoyl-CoA was largely converted into acetyl-CoA, and pent-2-enoyl-CoA into acetyl-CoA and propionyl-CoA. Penta-2,4-dienoyl-CoA was slowly converted into acetyl-CoA and acryloyl-CoA. 6. Penta-2,4-dienoyl-CoA, a unique metabolite of pent-4-enoate, was the only compound that specifically inhibited an enzyme of the beta-oxidation sequence, 3-oxoacyl-CoA thiolase. The formation of penta-2,4-dienoyl-CoA could explain the strong inhibition of fatty acid oxidation in intact mitochondria by pent-4-enoate.     

Pig liver pyruvate carboxylase. The reaction pathway for the carboxylation of pyruvate

1. The reaction pathway for the carboxylation of pyruvate, catalysed by pig liver pyruvate carboxylase, was studied in the presence of saturating concentrations of K(+) and acetyl-CoA. 2. Free Mg(2+) binds to the enzyme in an equilibrium fashion and remains bound during all further catalytic cycles. MgATP(2-) binds next, followed by HCO(3) (-) and then pyruvate. Oxaloacetate is released before the random release, at equilibrium, of P(i) and MgADP(-). 3. This reaction pathway is compared with the double displacement (Ping Pong) mechanisms that have previously been described for pyruvate carboxylases from other sources. The reaction pathway proposed for the pig liver enzyme is superior in that it shows no kinetic inconsistencies and satisfactorily explains the low rate of the ATP[unk][(32)P]P(i) equilibrium exchange reaction. 4. Values are presented for the stability constants of the magnesium complexes of ATP, ADP, acetyl-CoA, P(i), pyruvate and oxaloacetate.     

AMP-forming acetyl-CoA synthetases in Archaea show unexpected diversity in substrate utilization

Adenosine monophosphate (AMP)-forming acetyl-CoA synthetase (ACS; acetate:CoA ligase (AMP-forming), EC 6.2.1.1) is a key enzyme for conversion of acetate to acetyl-CoA, an essential intermediate at the junction of anabolic and catabolic pathways. Phylogenetic analysis of putative short and medium chain acyl-CoA synthetase sequences indicates that the ACSs form a distinct clade from other acyl-CoA synthetases. Within this clade, the archaeal ACSs are not monophyletic and fall into three groups composed of both bacterial and archaeal sequences. Kinetic analysis of two archaeal enzymes, an ACS from Methanothermobacter thermautotrophicus (designated as MT-ACS1) and an ACS from Archaeoglobus fulgidus (designated as AF-ACS2), revealed that these enzymes have very different properties. MT-ACS1 has nearly 11-fold higher affinity and 14-fold higher catalytic efficiency with acetate than with propionate, a property shared by most ACSs. However, AF-ACS2 has only 2.3-fold higher affinity and catalytic efficiency with acetate than with propionate. This enzyme has an affinity for propionate that is almost identical to that of MT-ACS1 for acetate and nearly tenfold higher than the affinity of MT-ACS1 for propionate. Furthermore, MT-ACS1 is limited to acetate and propionate as acyl substrates, whereas AF-ACS2 can also utilize longer straight and branched chain acyl substrates. Phylogenetic analysis, sequence alignment and structural modeling suggest a molecular basis for the altered substrate preference and expanded substrate range of AF-ACS2 versus MT-ACS1.     

Substrate stereochemistry of the biotin-dependent sodium pump glutaconyl-CoA decarboxylase from Acidaminococcus fermentans

The steric course of the decarboxylation of glutaconyl-CoA to crotonyl-CoA, catalysed by the biotin-dependent sodium pump glutaconyl-CoA decarboxylase from Acidaminococcus fermentans, was elucidated using the sequence: chiral acetate----citrate----glutamate----glutaconyl-CoA----crotonyl-CoA ----chiral acetate. Since glutaconyl-CoA or glutaconate labeled at C-4 was subjected to rapid chemical or enzymatic exchanges, glutamate was fermented to acetate by growing cells of A. fermentans. The analysis of the final chiral acetates gave following deviations from 50% in the fumarase exchange: + 13.8% starting with (R)-acetate and - 13.9% starting with (S)-acetate. The results demonstrated a retention of configuration during the decarboxylation. Thus glutaconyl-CoA decarboxylase adds to the list of biotin enzymes in which exclusive retention of configuration was observed. Glutaconate CoA-transferase from A. fermentans catalysed a 3H exchange of [2,4,4-3H]glutaconate with water when acetyl-CoA was present. At low concentration of acetyl-CoA (20 microM) the exchange ceased after exactly one atom 3H was released into the water, at high concentrations (1 mM) the exchange proceeded further. The apparent Km of acetyl-CoA in the exchange (1.1 microM) was 150 times smaller than that of the complete CoA transfer. It was concluded that either a mixed anhydride, between a carboxyl group of the enzyme and [2,4,4-3H]glutaconate, or enzyme-bound glutaconyl-CoA was the exchanging species.     

Selectivity of the yersiniabactin synthetase adenylation domain in the two-step process of amino acid activation and transfer to a holo-carrier protein domain

The adenylation (A) domain of the Yersinia pestis nonribosomal peptide synthetase that biosynthesizes the siderophore yersiniabactin (Ybt) activates three molecules of L-cysteine and covalently aminoacylates the phosphopantetheinyl (P-pant) thiols on three peptidyl carrier protein (PCP) domains embedded in the two synthetase subunits, two in cis (PCP1, PCP2) in subunit HMWP2 and one in trans (PCP3) in subunit HMWP1. This two-step process of activation and loading by the A domain is analogous to the operation of the aminoacyl-tRNA synthetases in ribosomal peptide synthesis. Adenylation domain specificity for the first step of reversible aminoacyl adenylate formation was assessed with the amino acid-dependent [(32)P]-PP(i)-ATP exchange assay to show that S-2-aminobutyrate and beta-chloro-L-alanine were alternate substrates. The second step of A domain catalysis, capture of the bound aminoacyl adenylate by the P-pant-SH of the PCP domains, was assayed both by catalytic release of PP(i) and by covalent aminoacylation of radiolabeled substrates on either the PCP1 fragment of HMWP2 or the PCP3-thioesterase double domain fragment of HMWP1. There was little selectivity for capture of each of the three adenylates by PCP3 in the second step, arguing against any hydrolytic proofreading of incorrect substrates by the A domain. The holo-PCP3 domain accelerated PP(i) release and catalytic turnover by 100-200-fold over the leak rate (<1 min(-1)) of aminoacyl adenylates into solution while PCP1 in trans had only about a 5-fold effect. Free pantetheine could capture cysteinyl adenylate with a 25-50-fold increase in k(cat) while CoA was 10-fold less effective. The K(m) of free pantetheine (30-50 mM) was 3 orders of magnitude larger than that of PCP3-TE (10-25 microM), indicating a net 10(4) greater catalytic efficiency for transfer to the P-pant arm of PCP3 by the Ybt synthetase A domain, relative to P-pant alone.     

Adenosine diphosphate sulphurylase activity in leaf tissue

1. A new method is described for the assay of ADP sulphurylase. The method involves sulphate-dependent [(32)P]P(i)-ADP exchange; the method is simpler, more sensitive and more direct than the method involving adenosine 5'-sulphatophosphate-dependent uptake of P(i). 2. ADP sulphurylase activity was demonstrated in crude extracts of leaf tissue from a range of plants. Crude spinach extract catalysed the sulphate-dependent synthesis of [(32)P]ADP from [(32)P]P(i); spinach extracts did not catalyse sulphate-dependent AMP-P(i), ADP-PP(i) or ATP-P(i) exchange under standard assay conditions. ADP sulphurylase activity in spinach leaf tissue was associated with chloroplasts and was liberated by sonication. 3. Some elementary kinetics of crude spinach leaf and purified yeast ADP sulphurylases in the standard assay are described; addition of Ba(2+) was necessary to minimize endogenous P(i)-ADP exchange of the yeast enzyme and crude extracts of winter-grown spinach. 4. Spinach leaf ADP sulphurylase was activated by Ba(2+) and Ca(2+); Mg(2+) was ineffective. The yeast enzyme was also activated by Ba(2+). The activity of both enzymes decreased with increasing ionic strength. 5. Purified yeast and spinach leaf ADP sulphurylases were sensitive to thiol-group reagents and fluoride. The pH optimum was 8. ATP inhibited sulphate-dependent P(i)-ADP exchange. Neither selenate nor molybdate inhibited sulphate-dependent P(i)-ADP exchange and crude spinach extracts did not catalyse selenate-dependent P(i)-ADP exchange. 6. The presence of ADP sulphurylase activity jeopardizes the enzymic synthesis of adenosine 5'-sulphatophosphate from ATP and sulphate with purified ATP sulphurylase and pyrophosphatase.     

Demonstration of carbon-carbon bond cleavage of acetyl coenzyme A by using isotopic exchange catalyzed by the CO dehydrogenase complex from acetate-grown Methanosarcina thermophila.

The purified nickel-containing CO dehydrogenase complex isolated from methanogenic Methanosarcina thermophila grown on acetate is able to catalyze the exchange of [1-14C] acetyl-coenzyme A (CoA) (carbonyl group) with 12CO as well as the exchange of [3'-32P]CoA with acetyl-CoA. Kinetic parameters for the carbonyl exchange have been determined: Km (acetyl-CoA) = 200 microM, Vmax = 15 min-1. CoA is a potent inhibitor of this exchange (Ki = 25 microM) and is formed under the assay conditions because of a slow but detectable acetyl-CoA hydrolase activity of the enzyme. Kinetic parameters for both exchanges are compared with those previously determined for the acetyl-CoA synthase/CO dehydrogenase from the acetogenic Clostridium thermoaceticum. Collectively, these results provide evidence for the postulated role of CO dehydrogenase as the key enzyme for acetyl-CoA degradation in acetotrophic bacteria.     

An enzyme-bound intermediate in the biosynthesis of 3-hydroxy-3-methylglutaryl-coenzyme A

1. Purified 3-hydroxy-3-methylglutaryl-CoA synthase from baker's yeast (free from acetoacetyl-CoA thiolase activity) catalysed an exchange of acetyl moiety between 3'-dephospho-CoA and CoA. The exchange rate was comparable with the overall velocity of synthesis of 3-hydroxy-3-methylglutaryl-CoA. 2. Acetyl-CoA reacted with the synthase, giving a rapid ;burst' release of CoA proportional in amount to the quantity of enzyme present. The ;burst' of CoA was released from acetyl-CoA, propionyl-CoA and succinyl-CoA (3-carboxypropionyl-CoA) but not from acetoacetyl-CoA, hexanoyl-CoA, dl-3-hydroxy-3-methylglutaryl-CoA, or other derivatives of glutaryl-CoA. 3. Incubation of 3-hydroxy-3-methylglutaryl-CoA synthase with [1-(14)C]acetyl-CoA yielded protein-bound acetyl groups. The K(eq.) for the acetylation was 1.2 at pH7.0 and 4 degrees C. Acetyl-labelled synthase was isolated free from [1-(14)C]acetyl-CoA by rapid gel filtration at pH6.1. The [1-(14)C]acetyl group was removed from the protein by treatment with hydroxylamine, CoA or acetoacetyl-CoA but not by acid. When CoA or acetoacetyl-CoA was present the radioactive product was [1-(14)C]acetyl-CoA or 3-hydroxy-3-methyl-[(14)C]glutaryl-CoA respectively. 4. The isolated [1-(14)C]acetyl-enzyme was slowly hydrolysed at pH6.1 and 4 degrees C with a first-order rate constant of 0.005min(-1). This rate could be stimulated either by raising the pH to 7.0 or by the addition of desulpho-CoA. 5. These properties are interpreted in terms of a mechanism in which 3-hydroxy-3-methyl-glutaryl-CoA synthase is acetylated by acetyl-CoA to give a stable acetyl-enzyme, which then condenses with acetoacetyl-CoA yielding a covalent derivative between 3-hydroxy-3-methylglutaryl-CoA and the enzyme which is then rapidly hydrolysed to free enzyme and product.     

Utilization of l-threonine by a species of Arthrobacter. A novel catabolic role for `aminoacetone synthase''

1. A species of Arthrobacter (designated Arthrobacter 9759) was isolated from soil by its ability to grow aerobically on l-threonine as sole source of carbon atoms, nitrogen atoms and energy; the organism also grew well on other sources of carbon atoms including glycine, but no growth was obtainable on aminoacetone or dl-1-aminopropan-2-ol. 2. During growth on threonine, (14)C from l-[U-(14)C]threonine was rapidly incorporated into glycine and citrate, and thereafter into serine, alanine, aspartate and glutamate. 3. With extracts of threonine-grown cells supplied with l-[U-(14)C]threonine, evidence was obtained of the NAD and CoA-dependent catabolism of l-threonine to produce acetyl-CoA plus glycine. Short-term incorporation studies in which [2-(14)C]acetate and [2-(14)C]glycine were supplied (a) to cultures growing on threonine, and (b) to extracts of threonine-grown cells, showed that the acetyl-CoA was metabolized via the tricarboxylic acid cycle and glyoxylate cycle whereas the glycine was converted into pyruvate via the folate-dependent ;serine pathway'. 4. The threonine-grown organism contained ;biosynthetic' threonine dehydratase and a potent NAD-linked l-threonine dehydrogenase but possessed no l-threonine aldolase activity. 5. Evidence was obtained that the acetyl-CoA and glycine produced from l-threonine had their immediate origin in the alpha-amino-beta-oxobutyrate formed by the threonine dehydrogenase; the CoA-dependent cleavage of this compound was catalysed by an alpha-amino-beta-oxobutyrate CoA-ligase, which was identified with ;aminoacetone synthase'. A continuous spectrophotometric assay of this enzyme was developed, and it was found to be inducibly synthesized only during growth on threonine and not during growth on acetate plus glycine. 6. By using a reconstituted mixture of separately purified l-threonine dehydrogenase and alpha-amino-beta-oxobutyrate CoA-ligase (i.e. ;aminoacetone synthase'), l-[U-(14)C]threonine was broken down to [(14)C]glycine plus [(14)C]acetyl-CoA (trapped as [(14)C]citrate). 7. There was no evidence of aminoacetone metabolism by Arthrobacter 9759 even though a small amount of this amino ketone appeared in the culture medium during growth on threonine.     

Identification of potential physiological activators of protein phosphatase 5

The protein serine/threonine phosphatase designated PP5 has little basal activity, and physiological activators of the enzyme have never been identified. Purified PP5 can, however, be activated by partial proteolysis or by the binding of supraphysiological concentrations of polyunsaturated long-chain fatty acids to its tetratricopeptide repeat (TPR) domain. To test whether activation of PP5 by polyunsaturated but not saturated fatty acids was an artifact of the lower solubility of saturated fatty acids, the effects of fatty acyl-CoA esters were examined. Saturated and unsaturated long-chain fatty acids are both freely water-soluble when esterified to CoA. Long-chain fatty acyl-CoA esters activated PP5 at physiological concentrations, with the saturated compounds being more effective. We investigated the effects of chain length and of the CoA moiety on PP5 activation. Chains of 16 carbons or more were required for optimal activation, with no activation observed below 10 carbons. On the basis of competition studies using acetyl-CoA, the function of the CoA moiety appeared to be to increase solubility of the fatty acyl moiety rather than to interact with a specific binding site. These data suggested that long-chain fatty acid-CoA esters might be physiological activators of PP5 and point to a potential link between fatty acid metabolism and signal transduction via this enzyme. Because heat shock protein 90 is also known to bind to the TPR domain of PP5 via its C-terminal domain (C90), we investigated its effect on PP5 activity. C90 activated the enzyme approximately 10-fold. Thus, we have identified two potential physiological activators of PP5.     

Short-chain fatty acid activation by acyl-coenzyme A synthetases requires SIR2 protein function in Salmonella enterica and Saccharomyces cerevisiae

SIR2 proteins have NAD(+)-dependent histone deacetylase activity, but no metabolic role has been assigned to any of these proteins. In Salmonella enterica, SIR2 function was required for activity of the acetyl-CoA synthetase (Acs) enzyme. A greater than two orders of magnitude increase in the specific activity of Acs enzyme synthesized by a sirtuin-deficient strain was measured after treatment with homogeneous S. enterica SIR2 protein. Human SIR2A and yeast SIR2 proteins restored growth of SIR2-deficient S. enterica on acetate and propionate, suggesting that eukaryotic cells may also use SIR2 proteins to control the synthesis of acetyl-CoA by the level of acetylation of acetyl-CoA synthetases. Consistent with this idea, growth of a quintuple sir2 hst1 hst2 hst3 hst4 mutant strain of the yeast Saccharomyces cerevisiae on acetate or propionate was severely impaired. The data suggest that the Hst3 and Hst4 proteins are the most important for allowing growth on these short-chain fatty acids.     

Escherichia coli coenzyme A-transferase: Kinetics,catalytic pathway and structure

The inducible acetyl-CoA:acetoacetate CoA-transferase of Escherichia coli catalyzes the transfer of CoA from acetyl-CoA to acetoacetate by a mechanism involving a covalent enzyme-CoA compound as a reaction intermediate. Acetyl-CoA + enzyme ? enzyme-CoA + Acetate Enzyme-CoA + acetoacetate ? acetoacetyl-CoA + enzyme These conclusions are based on the following data: 1) In the absence of acetoacetate, the maximal velocity of exchange of [¹⁴C]acetate into acetyl-CoA was comparable with maximal velocity of the complete reaction. 2) Incubation of the enzyme with NaBH₄ after preincubation with an acyl-CoA substrate inactivated the enzyme by reduction of a glutamate residue in the β subunit of the CoA-transferase to α-amino-δ-hydroxyvaleric acid. Given the susceptibility of thioesters to borohydride reduction, the enzyme-CoA bond is a γ-glutamyl thiolester 3) Following incubation of the enzyme with a fluorescent derivative of acetyl-CoA, 1,N⁶-ethenoacetyl-CoA, etheno-CoA was bound to the CoA-transferase. Free etheno-CoA did not bind to the enzyme.     

Differential effects of 2-oxo acids on pyruvate utilization and fatty acid synthesis in rat brain

1. The effects of 2-oxo-4-methylpentanoate, 2-oxo-3-methylbutanoate and 2-oxo-3-methylpentanoate on the activity of pyruvate dehydrogenase (EC 1.2.4.1), citrate synthase (EC 4.1.3.7), acetyl-CoA carboxylase, (EC 6.4.1.2) and fatty acid synthetase derived from the brains of 14-day-old rats were investigated. 2. The pyruvate dehydrogenase enzyme activity was competitively inhibited by 2-oxo-3-methylbutanoate with respect to pyruvate with a K(i) of 2.04mm but was unaffected by 2-oxo-4-methylpentanoate or 2-oxo-3-methylpentanoate. 3. The citrate synthase activity was inhibited competitively (with respect to acetyl-CoA) by 2-oxo-4-methylpentanoate (K(i)~7.2mm) and 2-oxo-3-methylbutanoate (K(i)~14.9mm) but not by 2-oxo-3-methylpentanoate. 4. The acetyl-CoA carboxylase activity was not inhibited significantly by any of the 2-oxo acids investigated. 5. The fatty acid synthetase activity was competitively inhibited (with respect to acetyl-CoA) by 2-oxo-4-methylpentanoate (K(i)~930mum) and 2-oxo-3-methylpentanoate (K(i)~3.45mm) but not by 2-oxo-3-methylbutanoate. 6. Preliminary experiments indicate that 2-oxo-4-methylpentanoate and 2-oxo-3-phenylpropionate (phenylpyruvate) significantly inhibit the ability of intact brain mitochondria from 14-day-old rats to oxidize pyruvate. 7. The results are discussed with reference to phenylketonuria and maple-syrup-urine disease. A biochemical mechanism is proposed to explain the characteristics of these diseases.     

Streptomyces coelicolor RedP and FabH enzymes, initiating undecylprodiginine and fatty acid biosynthesis, exhibit distinct acyl-CoA and malonyl-acyl carrier protein substrate specificities

RedP is proposed to initiate undecylprodiginine biosynthesis in Streptomyces coelicolor by condensing an acyl-CoA with malonyl-ACP and is homologous to FabH that catalyzes the same reaction for initiation of fatty acid biosynthesis. Herein, we report the substrate specificities of RedP and FabH from assays using pairings of two acyl-CoA substrates (acetyl-CoA and isobutyryl-CoA) and two malonyl-ACP substrates (malonyl-RedQ and malonyl-FabC). RedP activity was observed only with a pairing of acetyl-CoA and malonyl-RedQ, consistent with its proposed role in initiating the formation of acetyl-CoA-derived prodiginines. Malonyl-FabC is not a substrate for RedP, indicating that ACP specificity is one of the factors that permit a separation between prodiginine and fatty acid biosynthetic processes. FabH demonstrated greater catalytic efficiency for isobutyryl-CoA in comparison with acetyl-CoA using malonyl-FabC, consistent with the observation that in streptomycetes, a broad mixture of fatty acids is synthesized, with those derived from branched-chain acyl-CoA starter units predominating. Diminished FabH activity was also observed using malonyl-RedQ with the same preference for isobutyryl-CoA, completing biochemical and genetic evidence that in the absence of RedP this enzyme can produce branched-chain alkyl prodiginines.