Kinetic mechanism of the serine acetyltransferase from Haemophilus influenzae

The kinetic mechanism of serine acetyltransferase from Haemophilus influenzae was studied in both reaction directions. The enzyme catalyzes the conversion of acetyl CoA and L-serine to O-acetyl-L-serine (OAS) and coenzyme A (CoASH). In the direction of L-serine acetylation, an equilibrium ordered mechanism is assigned at pH 6.5. The initial velocity pattern in the absence of added inhibitors is best described by a series of lines converging on the ordinate when L-serine is varied at different fixed levels of acetyl CoA. The initial velocity pattern at pH 7.5 is also intersecting, but the lines are nearly parallel. Product inhibition by OAS is noncompetitive against acetyl CoA, while it is uncompetitive against L-serine. Product inhibition by L-serine in the reverse reaction direction is noncompetitive with respect to both OAS and CoASH. Glycine and S-methyl-L-cysteine (SMC) were used as dead-end analogs of L-serine and OAS, respectively. Glycine is competitive versus L-serine and uncompetitive versus acetyl CoA, while SMC is competitive against OAS and uncompetitive against CoASH. Desulfo-CoA was used as a dead-end analog of both acetyl CoA and CoASH, and is competitive versus both substrates in the direction of L-serine acetylation; while it is competitive against CoASH and noncompetitive against OAS in the direction of CoASH acetylation. All of the above kinetic parameters are consistent with those predicted for an ordered mechanism at pH 6.5 with the exception of the uncompetitive inhibition by OAS vs. serine. The latter inhibition pattern suggests combination of OAS with the central E:acetyl CoA:serine complex. Cysteine is known to regulate its own biosynthesis at the level of SAT. As a dead-end inhibitor, L-cysteine is competitive against both substrates in both reaction directions. These results are discussed in terms of the mechanism of regulation.     

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.     

Blasticidin S-producing Streptomyces morookaensis possesses an enzyme activity which hydrolyzes puromycin

Abstract Puromycin was inactivated without the presence of acetyl coenzyme A when incubated with extracts of blasticidin S-producing Streptomyces morookaensis . The two derivatives from puromycin, contained in the reaction mixture, were detected by thin-layer chromatography, purified by high performance liquid chromatography and analyzed for determination of the chemical structures by ¹H-nuclear magnetic resonance and positive-ion fast atom bombardment mass spectrometries. The analytical data revealed that puromycin was inactivated by the hydrolysis of amide linkage between the aminonucleoside and 0 -methyl-l-tyrosine moieties, suggesting that S. morookaensis possesses an enzyme activity which hydrolyzes puromycin.     

Hydroxy amino acid metabolism in Pseudomonas cepacia: role of L-serine deaminase in dissimilation of serine, glycine, and threonine.

Growth of Pseudomonas cepacia (P. multivorans) on serine depended upon induction of a previously undescribed L-serine deaminase distinct from threonine deaminase. Formation of the enzyme was induced during growth on serine, glycine, or threonine. The induction pattern reflected a role of the enzyme in catabolism of these three amino acids. Both threonine and glycine supported growth of serine auxotrophs and were presumably converted to serine and pyruvate in the course of their degradation. Mutant strains deficient in serine deaminase, or unable to use pyruvate as a carbon source, failed to utilize serine or glycine and grew poorly with threonine, whereas strains deficient in threonine dehydrogenase or alpha-amino beta-ketobutyrate:coenzyme A ligase (which together convert threonine to glycine and acetyl coenzyme A) failed to utilize threonine or derepress serine deaminase in the presence of this amino acid. The results confirm for the first time the role of alpha-amin beta-ketobutyrate:coenzyme A ligase in threonine degradation and indicate that threonine does not mimic serine as an inducer of serine deaminase.     

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.     

Metabolism of acetylene by Nocardia rhodochrous.

A Nocardia rhodochrous strain capable of utilizing acetylene as its sole source of carbon and energy exhibited slow growth on low concentrations of acetaldehyde. Resting cells incubated with acetylene formed a product identified as acetaldehyde, but attempts to demonstrate acetylene hydrase activity in cell-free extracts were unsuccessful. Acetaldehyde dehydrogenase in N. rhodochrous was found to be NAD+ linked and nonacylating, converting acetaldehyde to acetate. Specific activities of acetaldehyde dehydrogenase, acetothiokinase, and isocitrate lyase were enhanced in cells grown on acetylene and ethanol as compared with cells grown on alternate substrates. These results suggest that acetylene is catabolized via acetaldehyde to acetate and eventually to acetyl coenzyme A. Acetylene oxidation in N. rhodochrous appears to be constitutive and is not inhibited in the presence of either ethylene, nitrous oxide, or methane.     

Specific interactions of metal ions with Cys-Xaa-Cys unit inserted into the peptide sequence

In this work five peptides with Cys-Xaa-Cys motif were studied including Ac-Cys-Gly-Cys-NH(2), Ac-Cys-Pro-Cys-Pro-NH(2), their N-unprotected analogues and the N-terminal fragment of metallothionein-3, Met-Asp-Pro-Glu-Thr-Cys-Pro-Cys-Pro-NH(2). All these peptides were found to be very effective ligands for Ni(2+), Zn(2+) and Cd(2+) ions. Potentiometric and spectroscopic (UV-Vis, CD and MCD) studies have proved that sulfur atoms are critical donors for the metal ions coordination. The amide nitrogen may participate in the metal ion binding only in the case when Gly is adjacent to Cys residues. Ac-Cys-Gly-Cys-NH(2) may serve as a low molecular weight model for cluster A, which is a binding unit of nickel ion in acetyl coenzyme A synthase. This bifunctional enzyme from anaerobic microorganisms catalyzes the formation of acetyl coenzyme A from CO, a methyl group donated by the corrinoid-iron-sulfur protein and coenzyme A. Other peptides studied in this work were Ac-Cys-Pro-Cys-Pro-NH(2) and Met-Asp-Pro-Glu-Thr-Cys-Pro-Cys-NH(2) originating from metallothionein sequence. These motifs are characteristic for the sequence of cysteine rich metallothionein-3 (MT-3) called also neuronal growth inhibitory factor (GIF). Cys-Pro-Cys-Pro fragment of protein was demonstrated to be crucial for the inhibitory activity of the protein.     

Lipoic acid and diabetes—Part III: Metabolic role of acetyl dihydrolipoic acid

Rat liver lipoyl transacetylase catalyzes the formation of acetyl dihydrolipoic acid from acetyl coenzyme A and dihydrolipoic acid. In an earlier paper the formation of acetyl dihydrolipoic from pyruvate and dihydrolipoic acid catalyzed by pyruvate dehydrogenase has been reported. Acetyl dihydrolipoic acid is a substrate for citrate synthase, acetyl coenzyme A carboxylase and fatty acid synthetase. The V_max. for citrate synthase with acetyl dihydrolipoic acid was identical to acetyl coenzyme A (approximately 1 μmol citrate formed/min/mg protein) while the apparent K_m was approximately 4 times higher with acetyl dihydrolipoic acid as the substrate. This may be due to the fact that synthetic acetyl dihydrolipoic acid is a mixture of 4 possible isomers and only one of them may be the substrate for the enzymatic reaction. While dihydrolipoic acid can replace coenzyme A in the activation of succinate catalyzed by succinyl coenzyme A synthetase, the transfer of coenzyme A between succinate and acetoacetyl dihydrolipoic acid catalyzed by succinyl coenzyme A: 3 oxo-acid coenzyme A transferase does not occur.     

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.     

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.     

D-Serine inhibits serine palmitoyltransferase, the enzyme catalyzing the initial step of sphingolipid biosynthesis

Serine palmitoyltransferase (SPT), responsible for the initial step of sphingolipid biosynthesis, catalyzes condensation of palmitoyl coenzyme A and L-serine to produce 3-ketodihydrosphingosine (KDS). For determination of the stereochemical specificity of the amino acid substrate, a competition analysis of the production of [(3)H]KDS from L-[(3)H]serine was performed using purified SPT. D-Serine inhibited [(3)H]KDS production as effectively as non-radioactive L-serine, whereas neither D-alanine nor D-threonine showed any significant effect. Incubation of purified SPT with [palmitoyl 1-(14)C]palmitoyl coenzyme A and D-serine did not produce [(14)C]KDS, while the control incubation with L-serine did. These results suggest that D-serine competes with L-serine for the amino acid recognition site of SPT, but that D-serine is not utilized by this enzyme to produce KDS.     

Nonfunctional Tricarboxylic Acid Cycle and the Mechanism of Glutamate Biosynthesis in Acetobacter suboxydans

Acetobacter suboxydans does not contain an active tricarboxylic acid cycle, yet two pathways have been suggested for glutamate synthesis from acetate catalyzed by cell extracts: a partial tricarboxylic acid cycle following an initial condensation of oxalacetate and acetyl coenzyme A. and the citramalate-mesaconate pathway following an initial condensation of pyruvate and acetyl coenzyme A. To determine which pathway functions in growing cells, acetate-1-(14)C was added to a culture growing in minimal medium. After growth had ceased, cells were recovered and fractionated. Radioactive glutamate was isolated from the cellular protein fraction, and the position of the radioactive label was determined. Decarboxylation of the C5 carbon removed 100% of the radioactivity found in the purified glutamate fraction. These experiments establish that growing cells synthesize glutamate via a partial tricarboxylic acid cycle. Aspartate isolated from these hydrolysates was not radioactive, thus providing further evidence for the lack of a complete tricarboxylic acid cycle. When cell extracts were analyzed, activity of all tricarboxylic acid cycle enzymes, except succinate dehydrogenase, was demonstrated.     

Amino Acid Catabolism and Malic Enzyme in Differentiating Dictyostelium discoideum

Amino acids produced from protein degradation are the major energy source for differentiation and aging in Dictyostelium discoideum. Considering the reactions involved in the conversion of amino acids from an average protein into tricarboxylic acid cycle intermediates, a route from a cycle intermediate (probably malate) to acetyl coenzyme A is required for the complete utilization of amino acids. Citrate was isolated from cells pulse-labeled with (14)C-labeled amino acids and was cleaved with citrate lyase. When cells were pulse-labeled with [U-(14)C]-glutamate the specific radioactivity of the acetate and oxaloacetate portions of citrate were consistent with the conclusion that one-third of the carbon flowing through the tricarboxylic acid cycle is removed for the synthesis of acetyl coenzyme A. The data were also consistent with the patterns of carbon flux required to maintain steady-state levels of cycle intermediates in cells catabolizing amino acids. It is suggested that the malic enzyme (EC 1.1.1.40) catalyzes the synthesis of acetyl coenzyme A from malate and is responsible for the observed citrate labeling pattern. In cell extracts the activity of this enzyme increased markedly with the onset of differentiation. The properties of partially purified (40-fold) malic enzyme isolated at culmination indicated that the enzyme was allosteric and was positively affected by aspartate and glutamate. Thus, amino acid production from protein degradation would stimulate a reaction essential for the efficient utilization of these amino acids for energy.     

Amino acid and lactate catabolism in trimethylamine oxide respiration of Alteromonas putrefaciens NCMB 1735

The nonfermentative Alteromonas putrefaciens NCMB 1735 grew anaerobically in defined media with trimethylamine oxide as external electron acceptor. All amino acids tested, except taurine and those with a cyclic or aromatic side chain, were utilized during trimethylamine oxide-dependent anaerobic growth. Lactate, serine, and cysteine (which are easily converted to pyruvate) and glutamate and aspartate (which are easily converted to tricarboxylic acid cycle intermediates) were metabolized at the fastest rate. Growth with lactate as growth-limiting substrate gave rise to the formation of 40 mol% acetate, whereas serine and cysteine were nearly completely oxidized to CO2. Molar growth yields with the latter substrates were the same and were 50% higher than with lactate. This showed that more ATP was formed when acetyl coenzyme A entered the tricarboxylic acid cycle than when it was converted via acetyl phosphate to acetate. Also, growth with formate as substrate indicated that the reduction of trimethylamine oxide to trimethylamine was coupled with energy conservation by a respiratory mechanism.     

Amino acid and lactate catabolism in trimethylamine oxide respiration of Alteromonas putrefaciens NCMB 1735.

The nonfermentative Alteromonas putrefaciens NCMB 1735 grew anaerobically in defined media with trimethylamine oxide as external electron acceptor. All amino acids tested, except taurine and those with a cyclic or aromatic side chain, were utilized during trimethylamine oxide-dependent anaerobic growth. Lactate, serine, and cysteine (which are easily converted to pyruvate) and glutamate and aspartate (which are easily converted to tricarboxylic acid cycle intermediates) were metabolized at the fastest rate. Growth with lactate as growth-limiting substrate gave rise to the formation of 40 mol% acetate, whereas serine and cysteine were nearly completely oxidized to CO2. Molar growth yields with the latter substrates were the same and were 50% higher than with lactate. This showed that more ATP was formed when acetyl coenzyme A entered the tricarboxylic acid cycle than when it was converted via acetyl phosphate to acetate. Also, growth with formate as substrate indicated that the reduction of trimethylamine oxide to trimethylamine was coupled with energy conservation by a respiratory mechanism.     

Coenzyme M derivatives and their effects on methane formation from carbon dioxide and methanol by cell extracts of Methanosarcina barkeri.

Extracts of Methanosarcina barkeri reduced methanol and CO2 to CH4 in the presence of H2 and converted methanol stoichiometrically into CH4 and CO2 in the absence of H2. In dialyzed cell-free extracts these reactions were stimulated by 2-mercaptoethanesulfonic acid (coenzyme M) and some derivatives (acetyl and formylcoenzyme M and the oxidized form of coenzyme M), which could be converted to coenzyme M by enzyme systems present in the extracts. Methylcoenzyme M could not be used in these systems.     

Measurement of Serine Acetyltransferase Activity in Crude Plant Extracts by a Coupled Assay System Using Cysteine Synthase

Serine acetyltransferase (SATase) (EC 2.3.1.30 [EC] ) catalyzes theformation of Oacetyl-L-serine (OAS) from L-serine in the presenceof acetyl-CoA. A novel assay method was developed for measuringthis enzyme activity in extracts from plant tissues. The assayconsists of a coupled system in which the OAS formed is convertedto cysteine by the addition of cysteine synthase (CSase) (EC4.2.99.8 [EC] ). Cysteine thus formed is determined colorimetricallyand serves as a measure for SATase activity. This method israpid, simple and sensitive, and can be readily adapted formeasurement of SATase activity in crude tissue extracts or homogenates. (Received January 14, 1987; Accepted April 27, 1987)     

Partial purification and characterization of acetyl coenzyme A: taxa-4(20),11(12)-dien-5alpha-ol O-acetyl transferase that catalyzes the first acylation step of taxol biosynthesis

The acetylation of taxa-4(20),11(12)-dien-5alpha-ol is considered to be the third specific step of Taxol biosynthesis that precedes further hydroxylation of the taxane nucleus. An operationally soluble acetyl CoA:taxadienol-O-acetyl transferase was demonstrated in extracts of Taxus canadensis and Taxus cuspidata cells induced with methyl jasmonate to produce Taxol. The reaction was dependent on both cosubstrates and active enzyme, and the product of this acetyl transferase was identified by radiochromatographic and GC-MS analysis. Following determination of the time course of acetyl transferase appearance in induced cell cultures, the operationally soluble enzyme was partially purified by a combination of anion exchange, hydrophobic interaction, and affinity chromatography on immobilized coenzyme A resin. This acetyl transferase has a pI and pH optimum of 4.7 and 9.0, respectively, and a molecular weight of about 50,000 as determined by gel permeation chromatography. The enzyme shows high selectivity and high affinity for both cosubstrates, with Km values of 4.2 and 5.5 microM for taxadienol and acetyl CoA, respectively. The enzyme does not acetylate the more advanced Taxol precursors, 10-deacetylbaccatin III or baccatin III. This acetyl transferase is insensitive to monovalent and divalent metal ions, is only weakly inhibited by p-hydroxymercuribenzoate, N-ethylmaleimide, and coenzyme A, and resembles in general properties the few other O-acetyl transferases of higher plant origin that have been examined.     

ZmaR, a novel and widespread antibiotic resistance determinant that acetylates zwittermicin A.

ZmaR is a resistance determinant of unusual abundance in the environment and confers on gram-positive and gram-negative bacteria resistance to zwittermicin A, a novel broad-spectrum antibiotic produced by species of Bacillus. The ZmaR protein has no sequence similarity to proteins of known function; thus, the purpose of the present study was to determine the function of ZmaR in vitro. Cell extracts of E. coli containing zmaR inactivated zwittermicin A by covalent modification. Chemical analysis of inactivated zwittermicin A by 1H NMR, 13C NMR, and high- and low-resolution mass spectrometry demonstrated that the inactivated zwittermicin A was acetylated. Purified ZmaR protein inactivated zwittermicin A, and biochemical assays for acetyltransferase activity with [14C]acetyl coenzyme A demonstrated that ZmaR catalyzes the acetylation of zwittermicin A with acetyl coenzyme A as a donor group, suggesting that ZmaR may constitute a new class of acetyltransferases. Our results allow us to assign a biochemical function to a resistance protein that has no sequence similarity to proteins of known function, contributing fundamental knowledge to the fields of antibiotic resistance and protein function.     

The Utilization of Choline and Acetyl Coenzyme A for the Synthesis of Acetylcholine

Acetylcholine synthesis in rat brain synaptosomes was investigated with regard to the intracellular sources of its two precursors, acetyl coenzyme A and choline. Investigations with α-cyano-4-hydroxycinnamate, an inhibitor of mitochondrial pyruvate transport, indicated that pyruvate must be utilized by pyruvate dehydrogenase located in the mitochondria, rather than in the cytoplasm, as recently proposed. Evidence for a small, intracellular pool of choline available for acetylcholine synthesis was obtained under three experimental conditions. (1) Bromopyruvate competitively inhibited high-affinity choline transport, perhaps because of accumulation of intracellular choline which was not acetylated when acetyl coenzyme A production was blocked. (2) Choline that was accumulated under high-affinity transport conditions while acetyl coenzyme A production was impaired was subsequently acetylated when acetyl coenzyme A production was resumed. (3) Newly synthesized acetylcholine had a lower specific activity than that of choline in the medium. These results indicate that the acetyl coenzyme A that is used for the synthesis of acetylcholine is derived from mitochondrial pyruvate dehydrogenase and that there is a small pool of choline within cholinergic nerve endings available for acetylcholine synthesis, supporting the proposal that the high-affinity transport and acetylation of choline are kinetically coupled.