Random-order ternary complex reaction mechanism of serine acetyltransferase from Escherichia coli

Although serine acetyltransferase (SAT) from Escherichia coli is homologous with a number of bacterial enzymes that catalyze O-acetyl transfer by a sequential (ternary complex) mechanism, it has been suggested, from experiments with the nearly identical enzyme from Salmonella typhimurium, that the reaction could proceed via an acetyl-enzyme intermediate. To resolve the matter, the E. coli gene for SAT was overexpressed and the enzyme purified 13-fold to homogeneity. The results of a steady-state kinetic analysis of the forward reaction are diagnostic for a ternary complex mechanism, and the response of SAT to dead-end inhibitors indicates a random order for the addition of substrates. The linearity of primary double-reciprocal plots, in the presence and absence of dead-end inhibitors, argues that interconversion of ternary complexes is not significantly faster than kcat, whereas substrate inhibition by serine suggests that breakdown of the SAT.CoA binary complex is rate-determining. The results of equilibrium isotope exchange experiments, for both half-reactions, rule out a "ping-pong" mechanism involving an acetyl-enzyme intermediate, and a pre-steady-state kinetic analysis of the turnover of AcCoA supports such a conclusion. Kinetic data for the reverse reaction (acetylation of CoA by O-acetylserine) are also consistent with a steady-state random-order mechanism, wherein both the breakdown of the SAT*serine complex and the interconversion of ternary complexes are partially rate-determining.     

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.     

Purification and characterization of benzoate:coenzyme A ligase from Clarkia breweri

Benzoate:CoA ligase (BZL) was partially purified from flowers of the annual California plant Clarkia breweri. BZL catalyzes the formation of benzoyl-CoA and anthraniloyl-CoA, important intermediates for subsequent acyltransferase reactions in plant secondary metabolism. The native enzyme is active as a monomer with a molecular mass of approximately 59-64.5 kDa, and it has K(m) values of 45, 95, and 130 microM for benzoic acid, ATP, and CoA, respectively. BZL is most active in the pH range of 7.2-8.4, and its activity is strictly dependent on certain bivalent cations. BZL is an AMP-forming enzyme. Overall, its properties suggest that it is related to the family of CoA ligase enzymes that includes the plant enzyme 4-hydroxycinnamate:CoA ligase.     

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.     

Formation of palmityl-[13'-32P]coenzyme A from [gamma-32P]ATP in mitochondrial extracts of guinea pig liver.

Investigations of the incorporation of ³²P into acyl-coenzyme A (CoA) in incubation mixtures containing a soluble protein preparation derived from mitochondria, [γ-³²P]ATP, and palmityl-CoA have led to the discovery of an enzymatic activity which catalyzes the exchange of palmityl groups between molecules of CoA: CoA¹ + palmityl-CoA ? palmityl-CoA¹ + CoA. The preparation also contains dephospho-CoA kinase and palmityl-CoA thiolester hydrolase activities. The initial detection of the exchange reaction resulted from the formation of [3′-³²P]CoA via the dephospho-CoA kinase reaction with exogenous [γ-³²P]ATP. The described preparation of palmityl-[3′-³²P]CoA and palmityl-[³⁵S]CoA facilitated demonstration of the reversibility of the reaction and ruled out the possibility that the exchange of fragments of the CoA molecule mediated the observed incorporation. The reversible palmityl group exchange does not appear to be catalyzed by a previously described enzyme. None of the possible acyl group acceptors considered in these studies participated in the reaction as efficiently as CoA itself. The possibility is discussed that the exchange reaction may explain reports of an unknown lipid formed by an oligomycin-sensitive mitochondrial ATPase preparation.     

Acetyl coenzyme A: alpha-glucosaminide N-acetyltransferase. Evidence for a transmembrane acetylation mechanism

The lysosomal membrane enzyme acetyl-CoA: alpha-glucosaminide N-acetyltransferase catalyzes the transfer of an acetyl group from acetyl-CoA to terminal alpha-linked glucosamine residues of heparan sulfate. The reaction mechanism was examined using highly purified lysosomal membranes from rat liver. The reaction was followed by measuring the acetylation of a monosaccharide acetyl acceptor, glucosamine. The enzyme reaction was optimal above pH 5.5, and a 2-3-fold stimulation of activity was observed when the membranes were assayed in the presence of 0.1% taurodeoxycholate. Double reciprocal analysis and product inhibition studies indicated that the enzyme works by a Di-Iso Ping Pong Bi Bi mechanism. Further evidence to support this mechanism was provided by characterization of the enzyme half-reactions. Membranes incubated with acetyl-CoA and [3H]CoA were found to produce acetyl-[3H]CoA. This exchange was optimal at pH values above 7.0. Treating membranes with [3H] acetyl-CoA resulted in the formation of an acetyl-enzyme intermediate. The acetyl group could then be transferred to glucosamine, forming [3H]N-acetylglucosamine. The transfer of the acetyl group from the enzyme to glucosamine was optimal between pH 4 and 5. The results suggest that acetyl-CoA does not cross the lysosomal membrane. Instead, the enzyme is acetylated on the cytoplasmic side of the lysosome and the acetyl group is then transferred to the inside where it is used to acetylate heparan sulfate.     

Molecular and catalytic properties of the acetoacetyl-coenzyme A thiolase of Escherichia coli.

The acetoacetyl-CoA-thiolase, a product of the acetoacetate degradation operon (ato) was purified to homogeneity as judged by polyacrylamide-gel electrophoresis at pH 4.5, 7.0, and 8.3. The enzyme has a molecular weight of 166,000 and is composed of four identical subunits. The subunit molecular weight is 41,500. Histidine was the sole N-terminal amino acid detected by dansylation. The thiolase contains eight free sulhydryl residues and four intrachain disulfide bonds per mole. The ato thiolase catalyzes the CoA- dependent cleavage of acetoacetyl-CoA and the acetylation of acetyl-CoA to form acetoacetyl-CoA. The maximal velocity in the direction of acetoacetyl-CoA cleavage was 840 nmol min^? (enzyme unit)^?1 and the maximal velocity in the direction of acetoacetyl CoA formation was 38 nmol min^?1 (enzyme unit)^?1. Like other thiolases, the ato thiolase was inactivated by sulfhydryl reagents. The enzyme was protected from inactivation by sulfhydryl reagents in the presence of the acyl-CoA substrates, acetyl-CoA and acetoacetyl-CoA; however, no protection was obtained when the enzyme was incubated with the acetyl-CoA analog, acetylaminodesthio-CoA. Consistent with these results was the demonstration of an acetyl-enzyme compound when the thiolase was incubated with [1-¹⁴C]acetyl-CoA. The sensitivity of the acetyl-enzyme bond to borohydride reduction and the protection afforded by acyl-CoA substrates against enzyme inactivation by sulfhydryl reagents indicated that acetyl groups are bound to the enzyme by a thiolester bond.     

Enzyme-catalyzed acylation of homoserine: mechanistic characterization of the Haemophilus influenzae met2-encoded homoserine transacetylase

The first unique step in bacterial and plant methionine biosynthesis involves the acylation of the gamma-hydroxyl of homoserine. In Haemophilus influenzae, acylation is accomplished via an acetyl-CoA-dependent acetylation catalyzed by homoserine transacetylase. The activity of this enzyme regulates flux of homoserine into multiple biosynthetic pathways and, therefore, represents a critical control point for cell growth and viability. We have cloned homoserine transacetylase from H. influenzae and present the first detailed enzymatic study of this enzyme. Steady-state kinetic experiments demonstrate that the enzyme utilizes a ping-pong kinetic mechanism in which the acetyl group of acetyl-CoA is initially transferred to an enzyme nucleophile before subsequent transfer to homoserine to form the final product, O-acetylhomoserine. The maximal velocity and V/K(homoserine) were independent of pH over the range of values tested, while V/K(acetyl)(-)(CoA) was dependent upon the ionization state of a single group exhibiting a pK value of 8.6, which was required to be protonated. Solvent kinetic isotope effect studies yielded inverse effects of 0.75 on V and 0.74 on V/K(CoA) on the reverse reaction and effects of 1.2 on V and 1.7 on V/K(homoserine) on the forward reaction. Direct evidence for the formation of an acetyl-enzyme intermediate was obtained using rapid-quench labeling studies. On the basis of these observations, we propose a chemical mechanism for this important member of the acyltransferase family and contrast its mechanism with that of homoserine transsuccinylase.     

Nickel in subunit beta of the acetyl-CoA decarbonylase/synthase multienzyme complex in methanogens. Catalytic properties and evidence for a binuclear Ni-Ni site

The acetyl-CoA decarbonylase/synthase (ACDS) complex catalyzes the central reaction of acetyl C-C bond cleavage in methanogens growing on acetate and is also responsible for synthesis of acetyl units during growth on C-1 substrates. The ACDS beta subunit contains nickel and an Fe/S center and reacts with acetyl-CoA forming an acetyl-enzyme intermediate presumably directly involved in acetyl C-C bond activation. To investigate the role of nickel in this process two forms of the Methanosarcina thermophila beta subunit were overexpressed in anaerobically grown Escherichia coli. Both contained an Fe/S center but lacked nickel and were inactive in acetyl-enzyme formation in redox-dependent acetyltransferase assays. However, high activity developed during incubation with NiCl(2). The native and nickel-reconstituted proteins both contained iron and nickel in a 2:1 ratio, with insignificant levels of other metals, including copper. Binding of nickel elicited marked changes in the UV-visible spectrum, with intense charge transfer bands indicating multiple thiolate ligation to nickel. The kinetics of nickel incorporation matched the time course for enzyme activation. Other divalent metal ions could not substitute for nickel in yielding catalytic activity. Acetyl-CoA was formed in reactions with CoA, CO, and methylcobalamin, directly demonstrating C-C bond activation by the beta subunit in the absence of other ACDS subunits. Nickel was indispensable in this process too and was needed to form a characteristic EPR-detectable enzyme-carbonyl adduct in reactions with CO. In contrast to enzyme activation, EPR signal formation did not require addition of reducing agent, indicating indirect catalytic involvement of the paramagnetic species. Site-directed mutagenesis indicated that Cys-278 and Cys-280 coordinate nickel, with Cys-189 essential for Fe/S cluster formation. The results are consistent with an Ni(2)[Fe(4)S(4)] arrangement at the active site. A mechanism for C-C bond activation is proposed that includes a specific role for the Fe(4)S(4) center and accounts for the absolute requirement for nickel.     

Synthesis of acetyl coenzyme A from carbon monoxide, methyltetrahydrofolate, and coenzyme A by enzymes from Clostridium thermoaceticum

Two purified fractions from Clostridium thermoaceticum are shown to catalyze the following reaction: CO + CH3THF + CoA ATP leads to CH3COCoA + THF. The methyltetrahydrofolate (CH3THF) gives rise to the methyl group of the acetyl-coenzyme A (CoA) and the carbon monoxide (CO) and CoA to its carboxyl thio ester group. The role of ATP is unknown. One of the protein fractions (F2) is a methyltransferase, whereas the other fraction (F3) contains CO dehydrogenase and a methyl acceptor which is postulated to be a corrinoid enzyme. The methyltransferase catalyzes the transfer of the methyl group to the methyl acceptor, and the CO is converted to a formyl derivative by the CO dehydrogenase. By a mechanism that is as yet unknown, the formyl derivative in combination with CoA and the methyl of the methyl acceptor are converted to acetyl-CoA. It is also shown that fraction F3 catalyzes the reversible exchange of 14C from [1-14C]acetyl-CoA into 14CO and that ATP is required, but not the methyltransferase. It is proposed that these reactions are part of the mechanism which enables certain autotrophic bacteria to grow on CO. It is postulated that CH3THF is synthesized from CO and tetrahydrofolate which then, as described above, is converted to acetyl-CoA. The acetyl-CoA then serves as a precursor in other anabolic reactions. A similar autotropic pathway may occur in bacteria which grow on carbon dioxide and hydrogen.     

The kinetic mechanism and properties of the cytoplasmic acetoacetyl-coenzyme A thiolase from rat liver

1. Cytoplasmic acetoacetyl-CoA thiolase was highly purified in good yield from rat liver extracts. 2. Mg(2+) inhibits the rate of acetoacetyl-CoA thiolysis but not the rate of synthesis of acetoacetyl-CoA. Measurement of the velocity of thiolysis at varying Mg(2+) but fixed acetoacetyl-CoA concentrations gave evidence that the keto form of acetoacetyl-CoA is the true substrate. 3. Linear reciprocal plots of velocity of acetoacetyl-CoA synthesis against acetyl-CoA concentration in the presence or absence of desulpho-CoA (a competitive inhibitor) indicate that the kinetic mechanism is of the Ping Pong (Cleland, 1963) type involving an acetyl-enzyme covalent intermediate. In the presence of CoA the reciprocal plots are non-linear, becoming second order in acetyl-CoA (the Hill plot shows a slope of 1.7), but here this does not imply co-operative phenomena. 4. In the direction of acetoacetyl-CoA thiolysis CoA is a substrate inhibitor, competing with acetoacetyl-CoA, with a K(i) of 67mum. Linear reciprocal plots of initial velocity against concentration of mixtures of acetoacetyl-CoA plus CoA confirmed the Ping Pong mechanism for acetoacetyl-CoA thiolysis. This method of investigation also enabled the determination of all the kinetic constants without complication by substrate inhibition. When saturated with substrate the rate of acetoacetyl-CoA synthesis is 0.055 times the rate of acetoacetyl-CoA thiolysis. 5. Acetoacetyl-CoA thiolase was extremely susceptible to inhibition by an excess of iodoacetamide, but this inhibition was completely abolished after preincubation of the enzyme with a molar excess of acetoacetyl-CoA. This result was in keeping with the existence of an acetyl-enzyme. Acetyl-CoA, in whose presence the overall reaction could proceed, gave poor protection, presumably because of the continuous turnover of acetyl-enzyme in this case. 6. The kinetic mechanism of cytoplasmic thiolase is discussed in terms of its proposed role in steroid biosynthesis.     

Crystal structure of a substrate complex of Mycobacterium tuberculosis beta-ketoacyl-acyl carrier protein synthase III (FabH) with lauroyl-coenzyme A

Beta-ketoacyl-acyl carrier protein synthase III (FabH) catalyzes a two step reaction that initiates the pathway of fatty acid biosynthesis in plants and bacteria. In Mycobacterium tuberculosis, FabH catalyzes extension of lauroyl, myristoyl and palmitoyl groups from which cell wall mycolic acids of the bacterium are formed. The first step of the reaction is an acyl group transfer from acyl-coenzyme A to the active-site cysteine of the enzyme; the second step is acyl chain extension by two carbon atoms through Claisen condensation with malonyl-acyl carrier protein. We have previously determined the crystal structure of a type II, dissociated M.tuberculosis FabH, which catalyzes extension of lauroyl, myristoyl and palmitoyl groups. Here we describe the first long-chain Michaelis substrate complex of a FabH, that of lauroyl-coenzyme A with a catalytically disabled Cys-->Ala mutant of M.tuberculosis FabH. An elongated channel extending from the mutated active-site cysteine defines the acyl group binding locus that confers unique acyl substrate specificity on M.tuberculosis FabH. CoA lies in a second channel, bound primarily through interactions of its nucleotide group at the enzyme surface. The apparent weak association of CoA in this complex may play a role in the binding and dissociation of long chain acyl-CoA substrates and products and poses questions pertinent to the mechanism of this enzyme.     

2-Amino-3-ketobutyrate CoA ligase of Escherichia coli: stoichiometry of pyridoxal phosphate binding and location of the pyridoxyllysine peptide in the primary structure of the enzyme

Pure 2-amino-3-ketobutyrate CoA ligase from Escherichia coli, which catalyzes the cleavage/condensation reaction between 2-amino-3-ketobutyrate (the presumed product of the L-threonine dehydrogenase-catalyzed reaction) and glycine + acetyl-CoA, is a dimeric enzyme (Mr = 84,000) that requires pyridoxal 5'-phosphate as coenzyme for catalytic activity. Reduction of the hololigase with tritiated NaBH4 yields an inactive, radioactive enzyme adduct; acid hydrolysis of this adduct allowed for the isolation and identification of epsilon-N-pyridoxyllysine. Quantitative determinations established that 2 mol of pyridoxal 5'-phosphate are bound per mol of dimeric enzyme. After the inactive, tritiated enzyme adduct was digested with trypsin, a single radioactive peptide containing 23 amino acids was isolated and found to have the following primary structure: Val-Asp-Ile-Ile-Thr-Gly-Thr-Leu-Gly-Lys*-Ala-Leu-Gly-Gly-Ala-Ser-Gly-Gly -Tyr-Thr-Ala-Ala-Arg (where * = the lysine residue in azomethine linkage with pyridoxal 5'-phosphate). This peptide corresponds to residues 235-257 in the intact protein; 10 residues around the lysine residue have a high level of homology with a segment of the primary structure of 5-aminolevulinate synthase from chicken liver.     

The kinetic mechanism of 3-hydroxy-3-methylglutaryl-coenzyme A synthase from baker''s yeast

1. The effect of independent variation of both acetyl-CoA and acetoacetyl-CoA on the initial velocity at pH8.0 and pH8.9 gives results compatible with a sequential mechanism involving a modified enzyme tentatively identified as an acetyl-enzyme, resulting from the reaction with acetyl-CoA in the first step of a Ping Pong (Cleland, 1963a) reaction. 2. Acetoacetyl-CoA gives marked substrate inhibition that is competitive with acetyl-CoA. This suggests formation of a dead-end complex with the unacetylated enzyme and is in accord with the inhibition pattern given by 3-oxohexanoyl-CoA, an inactive analogue of acetoacetyl-CoA. 3. The inhibition pattern given by products of the reaction is compatible with the above mechanism. CoA gives mixed inhibition with respect to both substrates, whereas dl-3-hydroxy-3-methylglutaryl-CoA competes with acetyl-CoA but gives uncompetitive inhibition with respect to acetoacetyl-CoA. 4. 3-Hydroxy-3-methylglutaryl-CoA analogues lacking the 3-hydroxyl group are found to compete, like 3-hydroxy-3-methylglutaryl-CoA, with acetyl-CoA but have K(i) values ninefold higher, indicating the importance of the 3-hydroxyl group in the interaction. 5. A comparison of inhibition by CoA and desulpho-CoA at pH8.0 and pH8.9 shows that at the higher pH value a kinetically significant reversal of the formation of acetyl-enzyme can occur. 6. Acetyl-CoA homologues do not act as substrates and compete only with acetyl-CoA. A study of the variation of K(i) with acyl-chain length suggests the presence near the active centre of a hydrophobic region. 7. These results are discussed in terms of a kinetic mechanism in which there is only one CoA-binding site the specificity of which is altered by acetylation of the enzyme. 8. The rate of 3-hydroxy-3-methylglutaryl-CoA synthesis in yeast is calculated from the kinetic constants determined for purified 3-hydroxy-3-methylglutaryl-CoA synthase and from estimates of the physiological substrate concentrations. The rate of synthesis of 12nmol of 3-hydroxy-3-methylglutaryl-CoA/min per g wet wt. of yeast is still greater than the rate of utilization in spite of the extremely low (calculated) acetoacetyl-CoA concentration (1.8nm).     

Biochemical and molecular characterization of phenylacetate-coenzyme A ligase, an enzyme catalyzing the first step in aerobic metabolism of phenylacetic acid in Azoarcus evansii

Phenylacetate-coenzyme A ligase (PA-CoA ligase; AMP forming, EC 6.2. 1.30), the enzyme catalyzing the first step in the aerobic degradation of phenylacetate (PA) in Azoarcus evansii, has been purified and characterized. The gene (paaK) coding for this enzyme was cloned and sequenced. The enzyme catalyzes the reaction of PA with CoA and MgATP to yield phenylacetyl-CoA (PACoA) plus AMP plus PPi. The enzyme was specifically induced after aerobic growth in a chemically defined medium containing PA or phenylalanine (Phe) as the sole carbon source. Growth with 4-hydroxyphenylacetate, benzoate, adipate, or acetate did not induce the synthesis of this enzyme. This enzymatic activity was detected very early in the exponential phase of growth, and a maximal specific activity of 76 nmol min(-1) mg of cell protein(-1) was measured. After 117-fold purification to homogeneity, a specific activity of 48 micromol min(-1) mg of protein(-1) was achieved with a turnover number (catalytic constant) of 40 s(-1). The protein is a monomer of 52 kDa and shows high specificity towards PA; other aromatic or aliphatic acids were not used as substrates. The apparent K(m) values for PA, ATP, and CoA were 14, 60, and 45 microM, respectively. The PA-CoA ligase has an optimum pH of 8 to 8.5 and a pI of 6.3. The enzyme is labile and requires the presence of glycerol for stabilization. The N-terminal amino acid sequence of the purified protein showed no homology with other reported PA-CoA ligases. The gene encoding this enzyme is 1, 320 bp long and codes for a protein of 48.75 kDa (440 amino acids) which shows high similarity with other reported PA-CoA ligases. An amino acid consensus for an AMP binding motif (VX2SSGTTGXP) was identified. The biochemical and molecular characteristics of this enzyme are quite different from those of the isoenzyme catalyzing the same reaction under anaerobic conditions in the same bacterium.     

Purification and properties of benzoate-coenzyme A ligase, a Rhodopseudomonas palustris enzyme involved in the anaerobic degradation of benzoate.

A soluble benzoate-coenzyme A (CoA) ligase was purified from the phototrophic bacterium Rhodopseudomonas palustris. Synthesis of the enzyme was induced when cells were grown anaerobically in light with benzoate as the sole carbon source. Purification by chromatography successively on hydroxylapatite, phenyl-Sepharose, and hydroxylapatite yielded an electrophoretically homogeneous enzyme preparation with a specific activity of 25 mumol/min per mg of protein and a molecular weight of 60,000. The purified enzyme was insensitive to oxygen and catalyzed the Mg2+ ATP-dependent formation of acyl-CoA from carboxylate and free reduced CoA, with high specificity for benzoate and 2-fluorobenzoate. Apparent Km values of 0.6 to 2 microM for benzoate, 2 to 3 microM for ATP, and 90 to 120 microM for reduced CoA were determined. The reaction product, benzoyl-CoA, was an effective inhibitor of the ligase reaction. The kinetic properties of the enzyme match the kinetics of substrate uptake by whole cells and confirm a role for benzoate-CoA ligase in maintaining entry of benzoate into cells as well as in catalyzing the first step in the anaerobic degradation of benzoate by R. palustris.     

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.     

Fluorometric determination of phenylacetyl-CoA ligase from Pseudomonas putida: a very sensitive assay for a newly described enzyme

Phenylacetyl-CoA ligase (AMP-forming) from Pseudomonas putida is a newly described enzyme (Martinez-Blanco, H., Reglero, A., Rodriguez-Aparicio, L.B. and Luengo, J.M. (1990) J. Biol. Chem. 265, 7084-7090) specifically involved in the catabolism of phenylacetic acid. This enzyme catalyzes the formation of phenylacetyl-CoA in the presence of ATP, CoA, Mg2+ and phenylacetic acid. A rapid method of assaying this enzyme in partially purified preparations has been developed by coupling this reaction with adenylate kinase, pyruvate kinase and kinase and lactate dehydrogenase. The rate of phenylacetyl-CoA formation was measured indirectly by monitoring fluorometrically the NADH oxidation at 340 nm (excitation at 340 nm and analysis of the emitted light at 465 nm). The advantage of this method of assay over others (colorimetric, HPLC and spectrophotometric) is discussed.     

Mechanism of action of glutaryl-CoA and butyryl-CoA dehydrogenases. Purification of glutaryl-CoA dehydrogenase

Glutaryl-CoA dehydrogenase, a flavoprotein, catalyzes the reaction -OOCCH3CH2--CH2COSR (FAD leads to FADH2) leads to CH3CH = CHCOSR + CO2 (SR = CoA or pantetheine). With the isolated enzyme, a dye serves as the final electron acceptor. The enzyme from Pseudomonas fluorescens (ATCC 11250) has been purified to homogeneity. It was established with appropriate isotopic substitutions that the proton which is added to the gamma position of the product, subsequent to decarboxylation, is not derived from the solvent but is derived from the alpha position of the substrate. Under conditions where no net conversion of substrate occurs, i.e., in the absence of electron acceptor, the enzyme catalyzes the exchange of the beta hydrogen of the substrate with solvent protons. Butyryl-CoA dehydrogenase (M. elsedenii), which catalyzes an analogous reaction, catalyzes the exchange of both the alpha and beta hydrogens with solvent protons in the absence of electron acceptor. Glutaryl-CoA dehydrogenase and butyryl-CoA dehydrogenase are irreversibly inactivated by the substrate analogues 3-butynoylpantetheine and 3-pentynoylpantetheine. These inactivators do not form an adduct with the flavin and probably react with a nucleophile at the active site. Upon inactivation, the spectrum of the enzyme-bound flavin is essentially unchanged, and the flavin can be reduced by Na2S2O4. We suggest that inactivation involves intermediate allene formation. We proposed that these results support an oxidation mechanism for glutaryl-CoA dehydrogenase and butyryl-CoA dehydrogenase which is initiated by proton abstraction. With glutaryl-CoA dehydrogenase, the base, which abstracts the substrate alpha proton, is shielded from the solvent and is then used to protonate the carbanion (CH2--CH--CHCOSCoA) formed after oxidation and decarboxylation.     

Catalytic mechanism and inhibition of tRNA (uracil-5-)methyltransferase: evidence for covalent catalysis

tRNA (Ura-5-)methyltransferase catalyzes the transfer of a methyl group from S-adenosylmethionine (AdoMet) to the 5-carbon of a specific Urd residue in tRNA. This results in stoichiometric release of tritium from [5-3H]Urd-labeled substrate tRNA isolated from methyltransferase-deficient Escherichia coli. The enzyme also catalyzes an AdoMet-independent exchange reaction between [5-3H]-Urd-labeled substrate tRNA and protons of water at a rate that is about 1% that of the normal methylation reaction, but with identical stoichiometry. S-Adenosylhomocysteine inhibits the rate of the exchange reaction by 2-3-fold, whereas an analogue having the sulfur of AdoMet replaced by nitrogen accelerates the exchange reaction 9-fold. In the presence (but not absence) of AdoMet, 5-fluorouracil-substituted tRNA (FUra-tRNA) leads to the first-order inactivation of the enzyme. This is accompanied by the formation of a stable covalent complex containing the enzyme, FUra-tRNA, and the methyl group of AdoMet. A mechanism for catalysis is proposed that explains both the 5-H exchange reaction and the inhibition by FUra-tRNA: the enzyme forms a covalent Michael adduct with substrate or inhibitor tRNA by attack of a nucleophilic group of the enzyme at carbon 6 of the pyrimidine residue to be modified. As a result, an anion equivalent is generated at carbon 5 that is sufficiently reactive to be methylated by AdoMet. Preliminary experiments and precedents suggest that the nucleophilic catalyst of the enzyme is a thiol group of cysteine. The potent irreversible inhibition by FUra-tRNA suggests that a mechanism for the "RNA" effects of FUra may also involve irreversible inhibition of RNA-modifying enzymes.