Kinetic characterization of the [3'-32P]coenzyme A/acetyl coenzyme A exchange catalyzed by a three-subunit form of the carbon monoxide dehydrogenase/acetyl-CoA synthase from Clostridium thermoaceticum

The ability of acetyl coenzyme A synthesizing carbon monoxide dehydrogenase isolated from Clostridium thermoaceticum to catalyze the exchange of [3'-32P]coenzyme A with acetyl coenzyme A is studied. This exchange is found to have a rate exceeding that of the acetyl coenzyme A carbonyl exchange also catalyzed by CO dehydrogenase ([1-14C]acetyl coenzyme A + CO in equilibrium acetyl coenzyme A + 14CO). These two exchanges are diagnostic of the ability of CO dehydrogenase to synthesize acetyl coenzyme A from a methyl group, coenzyme A, and carbon monoxide. The kinetic parameters for the coenzyme A exchange have been determined: Km(acetyl coenzyme A) = 1500 microM, Km(coenzyme A) = 50 microM, and Vmax = 2.5 mumol min-1 mg-1. Propionyl coenzyme A is shown to be a substrate (Km approximately 5 mM) for the coenzyme A exchange, with a rate 1/15 that of acetyl coenzyme A, but is not a substrate for the carbonyl exchange. CO dehydrogenase capable of catalyzing both these two exchanges, and the oxidation of CO to CO2, is isolated as a complex of molecular weight 410,000 consisting of three proteins in an alpha 2 beta 2 gamma 2 stoichiometry. The proposed gamma subunit, not previously reported as part of CO dehydrogenase, copurifies with the enzyme and has the same molecular weight on sodium dodecyl sulfate-polyacrylamide gel electrophoresis as the disulfide reductase previously separated from CO dehydrogenase in a final chromatographic step.     

Crystal structure of mycothiol synthase (Rv0819) from Mycobacterium tuberculosis shows structural homology to the GNAT family of N-acetyltransferases

Mycothiol is the predominant low-molecular weight thiol produced by actinomycetes, including Mycobacterium tuberculosis. The last reaction in the biosynthetic pathway for mycothiol is catalyzed by mycothiol synthase (MshD), which acetylates the cysteinyl amine of cysteine-glucosamine-inositol (Cys-GlcN-Ins). The crystal structure of MshD was determined in the presence of coenzyme A and acetyl-CoA. MshD consists of two tandem-repeated domains, each exhibiting the Gcn5-related N-acetyltransferase (GNAT) fold. These two domains superimpose with a root-mean-square deviation of 1.7 A over 88 residues, and each was found to bind one molecule of coenzyme, although the binding sites are quite different. The C-terminal domain has a similar active site to many GNAT members in which the acetyl group of the coenzyme is presented to an open active site slot. However, acetyl-CoA bound to the N-terminal domain is buried, and is apparently not positioned to promote acetyl transfer. A modeled substrate complex indicates that Cys-GlcN-Ins would only fill a portion of a negatively charged channel located between the two domains. This is the first structure determined for an enzyme involved in the biosynthesis of mycothiol.     

Escherichia coli phosphoenolpyruvate carboxylase: studies on the mechanism of multiple allosteric interactions.

Some kinetic studies of the interactions between Escherichia coli phosphoenolpyruvate carboxylase (orthophosphate:oxaloacetate carboxylase (phosphorylating) EC 4.1.1.31) acetyl coenzyme A, fructose 1,6-bisphosphate, and aspartate were performed. Activation of the enzyme by fructose 1,6-bisphosphate is anomalous by comparison with acetyl coenzyme A in that it confers hysteretic properties on the enzyme. In the presence of both activators and aspartate, hysteresis is observed also, but the approach to optimum catalytic activity can be fit to an equation for a second-order reaction with respect to enzyme concentration. Since, however, hysteresis is not a result of any apparent association-dissociation reaction, the apparent fit to a second-order kinetic equation is probably not real but is the result of a multistep activation mechanism. Hysteresis is not eliminated by preincubation of the enzyme with fructose 1,6-bisphosphate, acetyl coenzyme A, or phosphoenolpyruvate singly or in any pair of combinations. Hysteresis is associated, therefore, with the slow conformation change from the inactive species to the active species under the influence of all three of those reactants. The enzyme complex resulting from the binding of each activator, including phosphoenolpyruvate, has an increased affinity for the other activators. A kinetic method for estimating the relative changes in affinity of these complexes for some of the other reactants is presented. At concentrations of the activators below their K_a, synergistic effects are evident, particularly in their ability to relieve aspartate inhibition. Aspartate inhibition is competitive with acetyl coenzyme A both in the absence and in the presence of low concentrations of fructose 1,6-bisphosphate. Increasing the concentrations of fructose 1,6-bisphosphate results in an increase in the apparent K_l for aspartate, suggesting that synergistic activation by fructose 1,6-bisphosphate is a result of the increased affinity of the fructose 1,6-bisphosphate-enzyme complex for acetyl coenzyme A, and a shift in the concentration of enzyme species away from the one(s) to which aspartate can bind most easily. In the presence of fructose 1,6-bisphosphate alone optimal activation can be achieved, but the concentrations required in vitro are high and suggest that fructose 1,6-bisphosphate alone does not function in that capacity physiologically, but primes the enzyme for more effective activation by acetyl coenzyme A and/or phosphoenolpyruvate.     

Interaction of the fluorescent analogue stearoyl-(1,N6)-etheno coenzyme A with chicken liver acetyl coenzyme A carboxylase

The interaction of stearoyl-(1,N6)-etheno coenzyme A (stearoyl-epsilon-CoA) with acetyl coenzyme A carboxylase was investigated by using fluorescence spectroscopy. The fluorescence emission of stearoyl-epsilon-CoA was partially quenched by acetyl coenzyme A carboxylase. Analysis of the data for dissociation constant (KD) and the stoichiometry of the interaction (n) gave values of 5.06 nM and 1.2, respectively, at pH 7.6 in 50 mM Tris-HCl and 25 degrees C. The KD value is comparable to the inhibition constant (Ki) obtained previously by others for the inhibition of rat liver acetyl coenzyme A carboxylase by long chain fatty acyl-CoAs. Citrate (which is known to polymerize and thus activate carboxylase) caused a partial quenching of the protein fluorescence of carboxylase, presumably due to polymerization of the enzyme. The quenching of the stearoyl-epsilon-CoA fluorescence caused by carboxylase as well as the inhibition of carboxylase activity by stearoyl-epsilon-CoA was reversed by citrate, but only in the presence of 6-O-methylglucose polysaccharide which forms a stable complex with fatty acyl-CoA. This shows that the stearoyl-epsilon-CoA bound to the enzyme is displaced by citrate only in the presence of an acceptor of fatty acyl-CoA. These results support the reciprocal relationship of citrate and fatty acyl-CoA in the regulation of acetyl coenzyme A carboxylase.     

Regulation at the phosphoenolpyruvate branchpoint in Azotobacter vinelandii: phosphoenolpyruvate carboxylase

Phosphoenolpyruvate carboxylase (EC 4.1.1.31) from Azotobacter vinelandii, like the corresponding enzyme from other organisms, is activated by acetyl coenzyme A and inhibited by l-aspartate. Both modifiers affect primarily the affinity of the enzyme for phosphoenolpyruvate. This is the first enzyme with a strictly anaplerotic (intermediate-replacing) function to be tested for response to the adenylate energy charge; it is entirely insensitive to variation in charge. The results suggest that carboxylation of phosphoenolpyruvate in this organism is controlled by negative feedback from aspartate and by the stimulatory effect of acetyl coenzyme A. The adenylate energy charge may be expected to affect the rate of this reaction indirectly through its effects on the concentrations of acetyl coenzyme A and l-aspartate.     

Crystal structure of serine acetyl transferase from Brucella abortus and its complex with coenzyme A

Brucella abortus is the major cause of premature foetal abortion in cattle, can be transmitted from cattle to humans, and is considered a powerful biological weapon. De novo cysteine biosynthesis is one of the essential pathways reported in bacteria, protozoa, and plants. Serine acetyltransferase (SAT) initiates this reaction by catalyzing the formation of O-acetylserine (OAS) using l-serine and acetyl coenzyme A as substrates. Here we report kinetic and crystallographic studies of this enzyme from B. abortus. The kinetic studies indicate that cysteine competitively inhibits the binding of serine to B. abortus SAT (BaSAT) and noncompetitively inhibits the binding of acetyl coenzyme A. The crystal structures of BaSAT in its apo state and in complex with coenzyme A (CoA) were determined to 1.96 Å and 1.87 Å resolution, respectively. BaSAT was observed as a trimer in a size exclusion column; however, it was seen as a hexamer in dynamic light scattering (DLS) studies and in the crystal structure, indicating it may exist in both states. The complex structure shows coenzyme A bound to the C-terminal region, making mostly hydrophobic contacts from the center of the active site extending up to the surface of the protein. There is no conformational difference in the enzyme between the apo and the complexed states, indicating lock and key binding and the absence of an induced fit mechanism.     

Steady-state kinetic analysis of phosphotransacetylase from Methanosarcina thermophila

Phosphotransacetylase (EC 2.3.1.8) catalyzes the reversible transfer of the acetyl group from acetyl phosphate to coenzyme A (CoA), forming acetyl-CoA and inorganic phosphate. A steady-state kinetic analysis of the phosphotransacetylase from Methanosarcina thermophila indicated that there is a ternary complex kinetic mechanism rather than a ping-pong kinetic mechanism. Additionally, inhibition patterns of products and a nonreactive substrate analog suggested that the substrates bind to the enzyme in a random order. Dynamic light scattering revealed that the enzyme is dimeric in solution.     

The crystal structure of spermidine/spermine N1-acetyltransferase in complex with spermine provides insights into substrate binding and catalysis

The enzyme spermidine/spermine N (1)-acetyltransferase (SSAT) catalyzes the transfer of acetyl groups from acetylcoenzyme A to spermidine and spermine, as part of a polyamine degradation pathway. This work describes the crystal structure of SSAT in complex with coenzyme A, with and without bound spermine. The complex with spermine provides a direct view of substrate binding by an SSAT and demonstrates structural plasticity near the active site of the enzyme. Associated water molecules bridge several of the intermolecular contacts between spermine and the enzyme and form a "proton wire" between the side chain of Glu92 and the N1 amine of spermine. A single water molecule can also be seen forming hydrogen bonds with the side chains of Glu92, Asp93, and the N4 amine of spermine. Site-directed mutation of Glu92 to glutamine had a detrimental effect on both substrate binding and catalysis and shifted the optimal pH for enzyme activity further into alkaline solution conditions, while mutation of Asp93 to asparagine affected both substrate binding and catalysis without changing the pH dependence of the enzyme. Considered together, the structural and kinetic data suggest that Glu92 functions as a catalytic base to drive an otherwise unfavorable deprotonation step at physiological pH.     

Acetyl Coenzyme A: Deacetylvindoline O-Acetyltransferase,A Novel Enzyme from Catharanthus

A new enzyme, Acetyl Coenzyme A: deacetylvindoline 0-acetyl transferase (EC 2.3.1. -) which catalyses the synthesis of vindoline from acetyl coenzyme A and deacetylvindoline was isolated from the soluble protein extract of Catharanthus roseus leaves and purified approximately 365-fold. The enzyme had an apparent pI of 4.6 upon chromatofocusing, an apparent molecular weight of 45,000 daltons and a pH optimum between 8.0 to 9.0. Dithiothreitol was essential to maintain enzyme activity.Substrate saturation studies of this enzyme resulted in Michaelis Menton kinetics giving Km values of 5.4 and 0.7µM respectively for acetyl coenzyme A and deacetylvindoline. Studies of the forward reaction demonstrated an absolute requirement for acetyl coenzyme A and deacetylvindoline derivatives containing a double bond at positions 6, 7, whereas the reverse reaction occurred only in the presence of free coenzyme A and vindoline derivatives containing the same double bond. The forward reaction was subject to product inhibition by coenzyme A with an apparent Ki of 8 µM, but was not inhibited by up to 2 mM vindoline. The rate of reaction could therefore be regulated by the level of free coenzyme A in the cell, unaffected by the accumulation of indole alkaloid product.It was suggested that this enzyme catalyses a late step in the biosynthesis of vindoline.     

Isolation and properties of N epsilon-hydroxylysine:acetyl coenzyme A N epsilon-transacetylase from Escherichia coli pABN11

The enzyme N epsilon-hydroxylysine acetylase has been isolated from Escherichia coli 294 carrying recombinant plasmid ABN11. Activity of the enzyme was followed by measurement of the rate of appearance of 2-nitro-5-thiobenzoate, the product of cleavage of 5,5'-dithiobis(2-nitrobenzoate) by free coenzyme A released from its acetyl derivative. The enzyme bound firmly to Reactive Blue 2-Sepharose CL-6B and was eluated with 1.5 M KCl. The protein gave a single band, corresponding to a Mr of 33,000, on polyacrylamide gel electrophoresis in sodium dodecyl sulfate. In contrast, gel filtration of the native enzyme gave a Mr of 150,000-200,000. A sequence analysis of the DNA at the junction of the first and second genes in the aerobactin operon, considered in conjunction with the N-terminal amino acid sequence of the isolated protein, enabled the conclusion that the acetylase is specified by the second gene in the complex. The enzyme transfers the acetyl moiety from acetyl coenzyme A to a variety of hydroxylamines, with N epsilon-hydroxylysine as the preferred substrate. In agreement with the results found by affinity chromatography, Coomassie Blue was observed to act as a potent inhibitor.     

Properties of a mutant Escherichia coli phosphoenolpyruvate carboxylase deficient in coregulation by intermediary metabolites.

Phosphoenolpyruvate carboxylase of Escherichia coli is activated by three different mechanisms: contiguous by acetyl coenzyme A, precursor by fructose 1,6-bisphosphate, and compensatory feedback by cytidine 5'-diphosphate (CDP). Even though each activator can interact independently with the enzyme, synergistic effects are observed with some combinations, namely, fructose 1,6-bisphosphate or CDP (coregulators), with acetyl coenzyme A. A mutant was isolated that has a phosphoenolpyruvate carboxylase which is refractory to activation by fructose, 1,6-bisphosphate and CDP. The mutant enzyme was shown to be active primarily as the dimer and to lack cooperativity in substrate binding. The binding of acetyl coenzyme A and substrate, however, was essentially the same as that of the wild-type enzyme. The mutant cells grew extremely slowly on glucose alone as the sole carbon source. The only defect in the mutant appeared to be the inability of this enzyme to be activated by the coregulators. These data are consistent with the thesis that coregulation by fructose 1,6-bisphosphate or CDP is an essential requirement for the activation in vivo of this enzyme.     

Crystal structure of the novel PaiA N-acetyltransferase from Thermoplasma acidophilum involved in the negative control of sporulation and degradative enzyme production

GCN5-related N-acetyltransferases (GNATs) are the most widely distributed acetyltransferase systems among all three domains of life. GNATs appear to be involved in several key processes, including microbial antibiotic resistance, compacting eukaryotic DNA, controlling gene expression, and protein synthesis. Here, we report the crystal structure of a putative GNAT Ta0374 from Thermoplasma acidophilum, a hyperacidophilic bacterium, that has been determined in an apo-form, in complex with its natural ligand (acetyl coenzyme A), and in complex with a product of reaction (coenzyme A) obtained by cocrystallization with spermidine. Sequence and structural analysis reveals that Ta0374 belongs to a novel protein family, PaiA, involved in the negative control of sporulation and degradative enzyme production. The crystal structure of Ta0374 confirms that it binds acetyl coenzyme A in a way similar to other GNATs and is capable of acetylating spermidine. Based on structural and docking analysis, it is expected that Glu53 and Tyr93 are key residues for recognizing spermidine. Additionally, we find that the purification His-Tag in the apo-form structure of Ta0374 prevents binding of acetyl coenzyme A in the crystal, though not in solution, and affects a chain-flip rotation of "motif A" which is the most conserved sequence among canonical acetyltransferases.     

Choline acetyltransferase structure reveals distribution of mutations that cause motor disorders

Choline acetyltransferase (ChAT) synthesizes acetylcholine in neurons and other cell types. Decreases in ChAT activity are associated with a number of disease states, and mutations in ChAT cause congenital neuromuscular disorders. The crystal structure of ChAT reported here shows the enzyme divided into two domains with the active site in a solvent accessible tunnel at the domain interface. A low-resolution view of the complex with one substrate, coenzyme A, defines its binding site and suggests an additional interaction not found in the related carnitine acetyltransferase. Also, the preference for choline over carnitine as an acetyl acceptor is seen to result from both electrostatic and steric blocks to carnitine binding at the active site. While half of the mutations that cause motor disorders are positioned to affect enzyme activity directly, the remaining changes are surprisingly distant from the active site and must exert indirect effects. The structure indicates how ChAT is regulated by phosphorylation and reveals an unusual pattern of basic surface patches that may mediate membrane association or macromolecular interactions.     

Reaction of the anionic acetylation agent methyl acetyl phosphate with D-3-hydroxybutyrate dehydrogenase

Methyl acetyl phosphate is a competitive inhibitor of the reduction of acetoacetate by D-3-hydroxybutyrate dehydrogenase. The material also irreversibly inactivates the enzyme. The kinetics of the inactivation are consistent with methyl acetyl phosphate acetylating the conjugate base of a hydrogen bond donor. Protection offered by a substrate analogue (methyl acetonylphosphonate) in the presence of coenzyme implicates reaction at the cationic active site. Reversible protection by the amino group reagent 2,3-dimethylmaleic anhydride suggests that methyl acetyl phosphate reacts with an amino group. Sulfhydryl reagents and acetyl phosphate, a poorer acetylating agent, do not inactivate the enzyme. The pH dependence of the inactivation suggests that the acetylation occurs at a site that has a pKa of 8.2. The utility of methyl acetyl phosphate and other acyl phosphate monoesters in reacting with lysines adjacent to cationic sites of enzymes, hemoglobin, and histones is noted.     

Purification and properties of citrate lyase from Escherichia coli

Citrate lyase (EC 4.1.3.6) has been purified from Escherichia coli and the homogeneity of the preparation established from the three-component subunits obtained on sodium dodecyl sulfate/polyacrylamide gel electrophoresis. The purified enzyme has a specific activity of 120 mumol min-1 mg-1 and requires optimally 10 mM Mg2+ and a pH of 8.0 for the cleavage reaction. The native enzyme is polydispersed in the ultracentrifuge and in polyacrylamide gel electrophoresis. The enzyme complex is composed of three different polypeptide chains of 85 000, 54 000, 32 000 daltons. An estimate of subunit stoichiometry indicates that 1 mol of the largest polypeptide chain is associated with 6 mol each of the smaller ones. The polypeptide subunits have been isolated in pure state and their biological functions characterize. The 54 000-dalton subunit functions as the acyltransferase alpha subunit catalyzing the formation of citryl coenzyme A from citrate in the presence of acetyl coenzyme A and ethylenediaminetetraacetic acid. The 32 000-dalton subunit functions as the acyllyase beta subunit catalyzing the cleavage of (3S)-citryl coenzyme A to oxal-acetate and acetyl coenzyme A. The 85 000-dalton subunit, which carries exclusively the prosthetic group components, functions as the acyl-carrier protein gamma subunit in the cleavage of citrate in the presence of mg2+ and the alpha and beta subunits. The presence of a large ACP subunit and the unusual stoichiometry of the different subunits distinguish the complex from other citrate lyases. A ligase which acetylates the deacetyl[citrate lyase] in the presence of acetate and ATP has ben shown to be present in the organism.(ABSTRACT TRUNCATED AT 250 WORDS)     

A nonchromatographic assay for expression of the chloramphenicol acetyltransferase gene in eucaryotic cells

A rapid procedure is described for assaying chloramphenicol acetyltransferase (CAT) enzyme activity following transfection of the CAT gene into eucaryotic cells. CAT enzyme activity in cell extracts catalyzes the transfer of [14C]acetyl groups from labeled acetyl coenzyme A to unlabeled chloramphenicol. Labeled reaction product is quantitated by liquid scintillation counting after extraction into ethyl acetate. The method is valid for use with transfected cell extracts only if the extracts are first heated to 65 degrees C to remove a factor which degrades acetyl coenzyme A. The revised procedure offers considerable advantages in speed and ease of performance over the chromatographic assay in current use.     

Choline acetyltransferase.

Acetylcholine is essential to neural function. It synthesis is catalyzed by choline acetyltransferase, the enzyme responsible for the acetylation of choline by acetyl coenzye A, a reaction favored slightly thermodymodynamically and not at all kinetically. An analytically pure enzyme still has not been obtained; however, method of purification have been greatly improved recently. Numerous inhibitors of the enzyme have been synthesized and their structure-action relationships examained. Evidence has been accumulated showing the essential involvement of an imidazole group in the active site of choline acetyltransferase. The literature regarding the controversial role to thiol groups in choline acetyltransferase is reviewed. Recently, derivatives of coenzyme A have been introduced as inhibitors of this enzyme and the specificity of coenzyme A binding has been examined. Possible mechanisms responsible for the control fo acetylcholine synthesis are discussed.     

Evidence from Fourier transform infrared spectroscopy for polarization of the carbonyl of oxaloacetate in the active site of citrate synthase

The infrared spectrum of oxaloacetate bound in the active site of citrate synthase has been measured in the binary complex and in the ternary complex with the acetyl coenzyme A (CoA) enolate analogue carboxymethyl-CoA. The carbonyl stretching frequency of oxaloacetate in binary and ternary complexes is found at 1697 cm-1, a shift of 21 cm-1 to lower frequency relative to that of the free ligand. The line widths of the carbonyl absorption in enzyme complexes differ from that of the free ligand, decreasing from a value of 20 cm-1 for the free ligand to 10 cm-1 in the binary complex and 7 cm-1 in the ternary complex with carboxymethyl-CoA. The integrated absorbance of the carbonyl absorption in these enzyme complexes is significantly increased over that of the free ligand at the same concentration, increasing approximately 2-fold in the binary complex and approximately 3-fold in the ternary complex. These results indicate strong polarization of the carbonyl bond in the enzyme-substrate complexes and suggest that ground-state destabilization is a major catalytic strategy of citrate synthase.     

Mutagen Stability of Alkylation-Sensitive Mutants of Bacillus subtilis

The phosphotransacetylase of Veillonella alcalescens catalyzes a reversible reaction with Michaelis-Menten kinetics for all substrates. The rate of the reverse reaction (the synthesis of acetyl coenzyme A from acetyl phosphate) was 6.5 times greater than the rate of the forward reaction (the synthesis of acetyl phosphate from acetyl coenzyme A). The apparent K(m) values determined for the forward reaction were 8.6 x 10(-6)m for acetyl coenzyme A and 9.3 x 10(-3)m for phosphate. In the reverse reaction, the K(m) values were 3.3 x 10(-4)m for coenzyme A and 5.9 x 10(-4)m for acetyl phosphate. The results of an analysis of the inhibition by end products in the forward and reverse directions were compatible with a random bi- bi- mechanism. The enzyme was inhibited by adenosine triphosphate and adenosine diphosphate but was not affected by reduced nicotinamide adenine dinucleotide or pyruvate. The inhibition by adenosine triphosphate was noncompetitive with respect to acetyl phosphate and competitive with respect to coenzyme A. MgCl(2) reversed the inhibition by adenosine triphosphate or adenosine diphosphate. The role of Mg(2+) and adenylates in the regulation of phosphotranscetylase activity is discussed.     

Phosphotransacetylase from Clostridium acidiurici

The phosphotransacetylase from Clostridium acidiurici has two properties not observed for this enzyme in other bacteria: (i) it requires a divalent metal for activity, and (ii) it is not subject to uncoupling by arsenate. The enzyme has been obtained in highly purified form, with a specific activity 500-fold higher than crude extracts. Ferrous or manganous ions are required for maximal activity, with Mn(2+) being 50 to 75% as effective as Fe(2+). The acetyl group can be transferred from acetyl phosphate to coenzyme A in 20 mm arsenate without a net decrease in high-energy acyl linkages. Likewise, H(32)PO(4) (2-) will exchange with acetyl-PO(4) (2-) in the presence of arsenate without loss of acetyl phosphate. This suggests that the active site on the enzyme is capable of discriminating between phosphate and arsenate while permitting the reversible transfer of acyl groups between CoA and phosphate.