Acetyl Donor Research Articles

The mechanism of N-terminal acetylation of proteins.

N alpha-acetylation is almost exclusively restricted to eukaryotic structural proteins. As a rule it is a post-initiational process, requiring the presence of the enzyme N alpha-acetyltransferase and the acetyl donor acetylcoenzyme A. N alpha-acetyltransferases appear to have a narrow substrate specificity, which is very similar for enzymes from different tissues and species. Amino acids predominantly present at the N terminus of N alpha-acetylated proteins are alanine, serine, and methionine. The occurrence of these residues is apparently a prerequisite for acetylation. The region following these amino acids is also important. If methionine is at the N terminus, the second position is always occupied by a strongly hydrophilic amino acid. Two- and three-dimensional structural characteristics of the protein do not seem to play a major role in N alpha-acetylation. Up to now the exact function for N alpha-acetylation is not known.

Characterization of the active site, substrate specificity and kinetic properties of acetyl-CoA:Arylamine N-acetyltransferase from pigeon liver

It could be demonstrated that a sulfhydryl group is involved in the catalysis of acetyl-CoA:arylamine N-acetyltransferase from pigeon liver (EC 2.3.1.5). From ping-pong kinetics it was concluded that there is a covalent acetyl-enzyme intermediate. The respective intermediate could be isolated and chemically characterized as a cysteinyl thioester. Electrophoretically homogeneous acetyl-CoA:acylamine N-acetyltransferase from pigeon liver was able to acetylate a broad variety of aromatic and aliphatic amines from different acetyldonors such as acetyl-CoA, p-nitroacetanilide and p-nitrophenylacetate. Apparent K m values were determined for a number of acetyl donors and acetyl acceptors. Additionally, K l values were evaluated for CoA, 3′,5′-ADP and AMP. Correlation studies of basicity of acceptor amines and acetylation rate demonstrated that there is a limit of the p K a value (about p K a = 1) where the covalently-bound acetyl-enzyme intermediate can still be saponified. Testing crude liver homogenates of several animals including turkey, duck, chicken, cow, pig, horse, sheep, carp, trout and herring the outstanding nature of the pigeon liver enzyme in acetylating very weakly basic amines could be demonstrated. It is shown that the enzyme is quite flexible concerning sterically different acceptor amines, because arylamines whose amino group was effected by large o-substituents could be quantitatively acetylated. After enzymatic acetylation of the first amino group, 1,2-phenylendiamine formed the heterocyclic compound 2-methylbenzimidazole by a spontaneous condensation reaction. There is evidence that with distinct amines formation of heterocyclic compounds may also occur in vivo.

Purification and physical-chemical properties of acetyl-CoA: Arylamine N-acetyltransferase from pigeon liver

Acetyl-CoA:arylamine N-acetyltransferase (EC 2.3.1.5) from pigeon liver was purified by protamine sulfate precipitation, ion exchange chromatography on DEAE-A-25 Sephadex, gel filtration on Sephadex G-75, amethopterin-AH-Sepharose 4B affinity chromatography, and finally, gel filtration on Sephadex G-100. The enzyme preparation was homogeneous as judged by ultracentrifugation studies, SDS-polyacrylamide gel electrophoresis and gel filtration. The N-terminal amino acid was detected to be histidine and the complete amino acid composition is reported. The enzyme contains one disulfide bridge and two cysteine residues/mol monomer. The isoelectric point was estimated to be 4.8. The molecular weight was determined to be 32 900 by high-speed sedimentation equilibrium analysis, 33 000 by Sephadex G-100 gel filtration and 31 600 by SDS-disc gel electrophoresis. The sedimentation coefficient from conventional sedimentation velocity runs was 3.1 S observed by ultraviolet optics. ‘Active enzyme centrifugation’ showed a sedimentation constant of 5.0 and 4.8 S for the purified enzyme and crude extract from pigeon liver, respectively, indicating that the enzyme forms a dimer under conditions of catalysis. It could be demonstrated that the inhibitor amethopterin was noncompetitive with respect to the acetyl donor and the acetyl acceptor. Acetyl-CoA:arylamine N-acetyltransferase was examined in different organs of pigeon. The enzyme was not inducible by 1,3-phenylenediamine and hexobarbital in vivo.

ChemInform Abstract: SYNTHESIS AND EVALUATION OF N‐(PHENYLALKYL)ACETOHYDROXAMIC ACIDS AS POTENTIAL SUBSTRATES FOR N‐ARYLHYDROXAMIC ACID N,O‐ACYLTRANSFERASE

AbstractDie Alko hole (I) ergeben über die entsprechenden Aldehyde die Aldoxime (II), deren Reduktion und anschließende Acetylierung zu den Hydroxamsäuren (III) führt.

The influence of GABA on the synthesis of N-acetylserotonin, melatonin, O-acetyl-5-hydroxytryptophol and O-acetyl-5-methoxytryptophol in the pineal gland of the male Wistar rat.

The influence of GABA on the synthesis of N-acetylserotonin, melatonin, O-acetyl-5-hydroxytryptophol and O-acetyl-5-methoxytryptophol has been investigated using different experimental procedures. It was demonstrated that when GABA and an acetyl donor were added to the incubation medium together, a significant increase in synthesis of the N-acetylated products occurred during the night. Moreover there was a large increase in N-acetylserotonin synthesis at 15(00) hrs although none was observed in the control experiments. However, when GABA was added 20 min before the acetyl donor, synthesis of the N-acetylated products was significantly less. The opposite effect was observed for the O-acetylated indoles. These results confirm the proposal by Ebadi et al. (1982) that GABA, like norepinephrine, may be a regulator of melatonin synthesis. As melatonin is implicated in the regulation of reproduction it may be that GABA is equally significant in this regulatory effect.

A Comparative Study of Citrate Efflux from Mitochondria of Oleaginous and Non‐oleaginous Yeasts

Citrate efflux from mitochondria of 10 different yeasts was investigated. Isolated mitochondria of all the yeasts show the possession of a carrier system for dicarboxylic and tricarboxylic anions, phosphate and pyruvate. The tricarboxylate carrier is also specific for l‐malate in all 10 yeasts. In citrate‐loaded mitochondria, citrate efflux and l‐malate uptake are directly proportional to the concentration of extramitochondrial l‐malate until the translocator becomes saturated (> 8 mM l‐malate in Candida curvata D). Rates of citrate efflux in the presence of l‐malate are linear for a period of 2 min and are approximately 2.5‐times greater in oleaginous than in non‐oleaginous yeasts. The malate‐citrate translocator has a significantly lower Km for l‐malate in oleaginous yeasts (∼ 4.0 mM) than in non‐oleaginous yeasts (∼ 8.4 mM). Both pyruvate and phosphate appear to stimulate the malate‐citrate exchange in mitochondria of C. curvata D. Oleginous yeast mitochondria contain 3—4‐times higher intramitochondrial citrate levels than non‐oleaginous yeast. In all these yeasts the enzymes for synthesis and metabolism of citrate were exclusively mitochondrial, the only exception being the possession of a cytosolic ATP; citrate lyase by oleaginous yeasts. In metabolically active mitochondria, once the malate‐citrate carrie is saturated with l‐malate, efflux of citrate is directly proportional to the concentration of extramitochondrial pyruvate (as acetyl donor). The rates of efflux of newly synthesised citrate from metabolically active mitochondria, in the presence of saturating l‐malate and pyruvate, are 6–8‐times greater in oleaginous yeasts and the citric‐acid‐producing yeast. The inhibition of aconitase by fluorocitrate produced a ninefold increase in citrate efflux from non‐oleaginous yeast mitochondria but only a slight increase in oleaginous yeasts. It is concluded that in fully functional yeast mitochondria, citrate efflux is limited by the amount of intra‐mitochondrial citrate made available to the citrate translocator for exchange.

Synthesis and evaluation of N-(phenylalkyl)acetohydroxamic acids as potential substrates for N-arylhydroxamic acid N,O-acyltransferase.

N-(4-Phenylcyclohexyl)acetohydroxamic acid and a series of N-(phenylalkyl)acetohydroxamic acids were synthesized and evaluated as substrates for partially purified rat and hamster hepatic arylhydroxamic acid N,O-acyltransferase systems (AHAT). The compounds were assayed for their abilities to function as acetyl donors in the AHAT-mediated transacetylation of 4-aminoazobenzene and for their abilities to participate in the AHAT-mediated conversion of N-arylhydroxylamines to electrophilic intermediates that form methylthio adducts upon reaction with N-acetylmethionine. None of the newly synthesized compounds displayed significant activity in either of the assays. The results of this study indicate that acetohydroxamic acids that have the nitrogen atom of the hydroxamic acid group attached directly to aliphatic or cycloalkyl groups are not likely to serve as substrates or inhibitors of AHAT.

Chemistry and biology of E. coli ribosomal protein L12.

E. coli ribosomal protein L12, because of its unique features, has been studied in more detail than perhaps any of the other ribosomal proteins. Unlike the other ribosomal proteins that are generally present in stoichiometric amounts, there are four copies of L12 per ribosome, some of which are acetylated on the N-terminal serine. The acetylated species, referred to as L7, has not been shown, as yet, to possess any different biological activity than L12. A specific enzyme that acetylates L12 to form L7, using acetyl-CoA as the acetyl donor, has been purified from E. coli extracts. L12 is also unique in that it does not contain cysteine, tryptophan, histidine, or tyrosine, is very acidic (pI: 4.85) and has a high content of ordered secondary structure (approximately 50%). The protein is normally found in solution as a dimer and also forms a tight complex with ribosomal protein L10. There are three methionine residues in L12, located in the N-terminal region of the protein, one or more of which are essential for biological activity. Oxidation of the methionines to methionine sulfoxide prevents dimer formation and inactivates the protein. The four copies of L12 are located in the crest region(s) of the 50S ribosomal subunit. There is good evidence that the soluble factors, such as IF-2, EF-Tu, EF-G and RF, interact with L12 on the ribosome during the process of protein synthesis. This interaction is essential for the proper functioning of each of the factors and for GTP hydrolysis associated with the individual partial reactions of protein synthesis. The L12 gene is located on an operon that contains the genes for L10 and beta beta' subunits of RNA polymerase at about 88 min on the bacterial chromosome. DNA-directed in vitro systems have been used to study the unique regulation of the expression of these genes. Autogenous regulation, translational control, and transcription attenuation are regulatory mechanisms that function to control the synthesis of these proteins.

35] N-acetyltransferase and arylhydroxamic acid acyltransferase

Publisher Summary This chapter presents procedures for the preparation of N -acetyltransferase and arylhydroxamic. N -acetyltransferase can be radioassayed using CoASAc as the acetyl donor. This method determines the extent to which the labeled acetyl group of CoASAc is transferred to the primary arylamine acceptor substrate to form an arylacetamide. N -Acetyltransferase can also be asssayed by calorimetric method using CoASAc as the acetyl donor. Acetylation is determined in this procedure from the decrease in the concentration of a primary aromatic amine in the incubation mixture; this decrease results from the transfer of the acetyl group of CoASAc to the acceptor amine. Arylhydroxamic acid N,O-acyltransferase assay determines the extent to which ring-labeled arylhydroxamic acids are incorporated into nucleic acid adducts as a consequence of the production of reactive N -acyloxyarylamines by N,O -acyl transfer. In enzyme purification, instability can be a major problem encountered in the purification of acyltransferases from the tissues of both the rabbit and rat, presumably caused by the oxidation of essential sulfhydryl groups. It is found useful to employ a chelating agent (pyrophosphate) as a buffer and a sulfhydryl protective agent (dithiothreitol) during purification.

Formula omitted] synthesis of [formula omitted]-pipecolate from [formula omitted]-lysine: Inconsistent with [formula omitted] as an obligatory intermediate

In vitro synthesis of 14 C- L - pipecolic acid from L -[ U- 14 C] lysine was demonstrated for the first time in the rat tissues. Acetyl donor was not required for this synthesis. No labeling of ϵ-N-acetyl- L-lysine was detected during 14C-pipecolate synthesis. When ϵ-N-acetyl- L-[U- 14C]lysine was substrate, large quantities of 14C-lysine but only very small quantities of 14C-pipecolate were detected, indicating the synthesis of L - pipecolate from ϵ-N-acetyl- L-lysine to be by way of L - lysine . Although the present data are inconsistent with the hypothesis that removal of α-NH 2 group from L - lysine in preparation for L - pipecolate formation requires ϵ-N-substitution, the involvement of an enzyme-bound ϵ-N-substituted L - lysine in this pathway cannot be ruled out.

Studies on the metabolic pathway of the acetyl group for acetylcholine synthesis

Glucose and pyruvate are the most effective precursors of the acetyl moiety of acetylcholine in mammalian brain; the metabolic intermediates between pyruvate and acetylcholine, however, are unknown. The following data suggest that citrate is not the sole intermediate of the acetyl group for acetylcholine synthesis in rat brain slices or synaptosomes: (1) 2.5 mM (−)-hydroxycitrate decreased acetylcholine synthesis from [U- 14C]glucose by only 25 per cent; (2) inhibition of citrate transport out of mitochondria by n-butylmalonate or 1,2,3-benzenetricarboxylate variably affected acetylcholine synthesis; and (3) high concentrations of nonradioactive citrate decreased the synthesis of acetylcholine but did not decrease the specific activity of the acetylcholine synthesized from [U- 14C]glucose. even though the uptake of citrate into the synaptosomes under these experimental conditions was approximately five times greater than the uptake of glucose. Other possible acetyl donors altered acetylcholine synthesis. Acetylcarnitine stimulated synthesis in brain slices, and carnitine stimulated synthesis by synaptosomes.The specificactivity of the acetylcholine synthesized from [U- 14Cglucose by synaptosomes was decreased by N-acetyl- l-aspartate (10mM), acetyl CoA (1 mM), and acetyl phosphate (10mM) which is consistent with these compounds acting as direct acetyl donors. Acetate (10 mM) did not affect either the amount or specific activity of the acetylcholine synthesized. Further evidence of compartmentation of cytoplasmic acetyl CoA is presented. The cytoplasmic acetyl CoA for acetylcholine synthesis is distinguishable from the cytoplasmic acetyl CoA for lipid synthesis. (−)-Hydroxycitrate inhibited acetylcholine synthesis without inhibiting lipid synthesis from [U- 14C]glucose. However, when 3-hydroxy[3- 14C]butyrate was used as substrate, (−)-hydroxycitrate inhibited incorporation into lipids twice as much as incorporation into acetylcholine. [U- 14C]Glucose metabolism by infant brain slices was more sensitive than adult brain slices to (−)-hydroxycitrate. However, the response to the other compounds which interfere with citrate metabolism was similar in slices from adult and infant brains.

Role of AMP in regulation of the citric acid cycle in mitochondria from baker's yeast

The role of AMP in regulation of the citric acid cycle has been investigated in mitochondria prepared from mechanically disrupted baker's yeast ( Saccharomyces cerevisiae). Addition of citric acid cycle intermediates and the acetyl donors: pyruvate, acetaldehyde and acetate in micromolar concentrations caused discrete and reproducible oxygen uptake with a stoicheiometric relationship to substrate amount and metabolite formation, which allowed calculations of the extent of oxidation separately for acetyl donors and auxiliary substrate. AMP increased the rate of citrate oxidation in the mitochondrial suspensions to about the same level as found for oxidation of succinate and 2-oxoglutarate. The total oxygen consumption and malate formation agreed with oxidation through the citric acid cycle also for citrate, indicating insignificant contamination with peroxisomes and enzymes of the glyoxylate pathway. In the absence of AMP, addition of acetyl donor plus auxiliary substrate (malate for pyruvate and 2-oxoglutarate for acetaldehyde and acetate) led to citrate accumulation and an oxygen consumption which indicated that the auxiliary substrate had been utilized only once for citrate formation. Malate, succinate and citrate were unable to support acetate and acetaldehyde oxidation to the same extent as found with malate supported pyruvate oxidation. AMP increased the rate of acetyl donor oxidation, reduced citrate accumulation almost to zero and gave rise to complete oxidation of acetyl donors at molar acetyl donor/auxiliary substrate ratios up to 3. Complete oxidation of acetate was obtained also with citrate as auxiliary substrate, but not with malate or succinate. Half maximal stimulation of the rate of citrate and of acetate oxidation was acetate oxidation in the absence of AMP despite the concomitant oxidation 2-oxoglutarate. The reason for this failure is not clear. The low rate of citrate (isocitrate) oxidation might create conditions unfavourable for acetate activation e.g., by allowing side reactions to compete successfully for intermediates in substrate phosphorylation at the expense of reactions leading to acetate activation. In this case the AMP effects on acetate oxidation could be explained solely as a result of activation of the NAD-dependent isocitrate dehydrogenase. The results obtained in the present investigation are in general agreement with such a “one site” suggestion, but the results are on the other hand difficult to reconcile with the role of AMP in acetate activation considering that the activating system and isocitrate dehydrogenase are both localized at or inside the inner mitochondrial membrane. A more detailed study of acetate activation under the conditions employed in the present study seems therefore required.

Hémisynthèse d'antibiotiques aminoglycosidiques: I. - Mise au point d'un réacteur enzymatique à cofacteur

In order to produce specifically N-monoalkylated derivatives of aminoglycoside antibiotics of potential therapeutic values, we have developed an enzymatic reactor. This system uses the aminoglycoside acetyltransferase as catalyst and acetylcoenzyme A as acetyl donor. The immobilization of one aminoglycoside acetyltransferase on different resins has been studied. The coreticulation of this enzyme on DEAE cellulose in the presence of glutaraldehyde gives rise to an enzymatic resin of high efficiency. On the other hand, we have also studied the acetylation of coenzyme A in a simple manner. Acetylation occurs in a quantitative yield when the reaction is performed in the presence of polyvinyl-4 pyridine/divinylbenzene 2 per cent. These conclusions enabled to develop two types of acetylating reactors which give rise without purification to 3-acetyl gentamicin.

The synthesis of acetylcholine from acetyl-CoA, acetyl-dephospho-CoA and acetylpantetheine phosphate by choline acetyltransferase.

Abstract— The synthesis of ACh by choline acetyltransferase (ChAc) has been examined using acetyl‐CoA, acetyl‐dephospho‐CoA and acetylpantetheine phosphate. At pH 7.5 Km values of 25.7 μm for acetyl‐CoA, 54.8 μm for acetyl‐dephospho‐CoA and 382 μm for acetylpantetheine phosphate were obtained and are similar to those at pH 6.0. This indicates that the 3‐phosphate may not be required for binding the substrate to the enzyme unlike carnitine acetyltransferase.Inhibitor constants (Ki) for CoA, dephospho‐CoA and pantetheine phosphate were also measured and when considered with the Km values obtained for the acetyl derivatives it is concluded that acetyl‐dephospho‐CoA could be a successful acetyl donor in the synthesis of ACh.Acetyl‐dephospho‐CoA was found to be less satisfactory as a substrate for citrate synthase.

Enzymatic N-acetylation of indolealkylamines by brain homogenates of the honeybee, Apis mellifera

Brain homogenates of the honey-bee, Apis mellifera, have been found to possess enzymes capable of catalysing the N-acetylation of tryptamine and 5-hydroxytryptamine with acetyl coenzyme A as the acetyl donor. The K m of the N-acetylation of tryptamine was 5·0 × 10 −7 M at pH 7·0 and 33°C. Evidence was obtained that the indolealkylamines, tryptamine, and 5-hydroxytryptamine, are not oxidized by monoamine oxidase (MAO) as is commonly considered to be a major catabolic route in vertebrate animals. The assay of Wurtman and Axelrod, reportedly specific for monoamine oxidase activity, will not distinguish between oxidation by MAO and N-acetylation of tryptamine and so should not be used to assay for MAO activity in insect tissues without careful identification of the products of the reaction. Implications of N-acetylation of indoleaklamines are discussed in relation to the neurotransmitter problem.

Mechanism of enzymic acetylation of des-acetyl citrate lyase.

A new enzyme, acetate:SH-[acyl-carrier-protein] enzyme ligase (AMP), has been purified about 30-fold from cell-free extracts of Klebsiella aerogenes. The enzyme, in the presence of acetate and ATP, catalyzes the acetylation of enzymically inactive des-acetyl citrate lyase to enzymically active citrate lyase (EC 4.1.3.6). Acetate and ATP could be replaced by acetyl adenylate. Acetyl-CoA can act as acetyl donor in this activation only if trace amounts of adenine nucleotides and auxiliary enzymes are present. These allow formation of acetyl adenylate in acetate- and ATP-generating reactions.

S-Acetyl phosphopantetheine: Deacetyl citrate lyase S-acetyl transferase from Klebsiella aerogenes

An enzyme has been partially purified from Klebsiella aerogenes which transfers an acetyl group from S-acetyl phosphopantetheine to deacetyl citrate lyase. This converts the deacetyl citrate lyase which has no enzyme activity, to citrate lyase, the active enzyme. A variety of other acetyl thioesters including acetyl CoA did not serve as acetyl donors.

Protein Modifications by Activated Carcinogens: I. THE ACETYLATION OF RIBONUCLEASE BY N-ACETOXY-2-FLUORENYLACETAMIDE

Abstract The postulated fragmentation of RNase by N-acetoxy-2-fluorenylacetamide (N-acetoxy-2-FAA), an activated metabolite of the carcinogen, N-hydroxy-2-fluorenylacetamide, has been investigated. The interaction of N-acetoxy-2-FAA with RNase resulted in the formation of two new proteins separable from RNase by electrophoresis and ion exchange chromatography. These proteins contained no new NH2-terminal amino acids, and their amino acid composition was identical with that of native RNase, indicating that the protein had not been modified by cleavage of the peptide chain. The alternative possibility that the formation of the additional proteins was due to a decrease in the positive charge of RNase was examined by the determination of the incorporation of 3H and 14C from N-[3H]acetoxy-[9-14C]2-FAA into RNase. The ratio of bound 3H to bound 14C, which measures the relative extent of acetylation and arylamidation in the modified proteins, suggested that acetylation rather than arylamidation was the predominant reaction modifying RNase. The conclusion that the acetyl group of N-[3H]-acetoxy-[9-14C]2-FAA had been transferred to RNase was confirmed (a) by the recovery of [3H]acetic acid from the hydrolysates of the modified protein and (b) by the isolation of e-N-acetyl-l-lysine from enzymatic hydrolysates of the modified protein. The isolation of the N-acetylated amino acid accounted in part for the decrease in the positive charge of RNase after reaction with N-acetoxy-2-FAA. The results indicate that N-acetoxy-2-FAA is an acetyl donor and modifies proteins primarily by acetylation rather than arylamidation.

Formation of electrophilic N-acetoxyarylamines in cytosols from rat mammary gland and other tissues by trans-acetylation from the carcinogen N-hydroxy-4-acetylaminobiphenyl

The 105 000 × g supernatant fractions of various rat tissues catalyze the transfer of the N-acetyl group of certain carcinogenic aromatic acethydroxamic acids to the O atom of aromatic hydroxylamines. The resulting N-acetoxyhydroxylamines are strongly electrophilic and have been detected and analyzed through their reaction with N-acetylmethionine to yield methylmercaptoaminoarenes. Of the rat tissues studied the liver had the highest activity; kidney and small intestinal mucosa were about 15–20% as active. The transacetylase activities of these tissues were similar with respect to their ability to use either N-hydroxy-2-acetylaminofluorene ( N-hydroxy-AAF or N-hydroxy-4-acetylaminobiphenyl ( N-hydroxy-AABP) as acetyl donors, their stability on storage at 2–3°C, and their elution patterns from Sephadex G-100 columns. Low transacetylase activity was found in spleen and muscle. Mammary tissue from 16–21 day pregnant rats had 20% of the transacetylase activity of rat liver when N-hydroxy-AABP was used as acetyl donor and N-hydroxy-4-aminobiphenyl ( N-hydroxy-ABP) was the acetyl acceptor. This enzyme system from mammary tissue did not utilize the fluorene derivatives as either acetyl donor or acetyl acceptor, was much more labile than the liver, kidney, or intestinal mucosa systems, and had a pH optimum at 7.5, as compared to pH 6.8 for liver. The mammary tissue system was similar to the hepatic system in being inhibited by sulfhydryl reagents; it required a source of reduced pyridine nucleotides for maximum activity.

Electrophilic N-acetoxyaminoarenes derived from carcinogenic N-hydroxy- N-acetylaminoarenes by enzymatic deacetylation and transacetylation in liver

• Incubation of N-hydroxy-2-acetylaminofluorene or N-hydroxy-4-acetylaminobiphenyl with N-hydroxy-2-aminofluorene or N-hydroxy-4-aminobiphenyl, rat liver cytosol, and N-acetylmethionine yields 3-methylmercapto-2-aminofluorene or 3-methylmercapto-4-aminobiphenyl. The apparent mechanism is a transacetylation from the hydroxamic acid to the hydroxylamine to yield an N-acetoxyaminoarene. The latter compound then reacts with N-acetylmethionine to yield a sulfonium derivative which decomposes to the methylmercaptoaminoarene. This formulation of the mechanism is supported by the finding that the same methylmercaptoaminoarenes are formed on incubation at neutrality of the hydroxylamines with acetic anhydride and N-acetylmethionine. The same reactions, both with the enzymatic system and with acetic anhydride, have been observed with certain other arylhydroxylamines. In each case with the acetic anhydride system and in two cases with the enzymatic system the products have been isolated by thick-layer chromatography and characterized by their mass spectra. Substitution of guanosine as the nucleophilic reactant in the transacetylase system with N-hydroxy-2-acetylaminofluorene and N-hydroxy-2-aminofluorene resulted in the formation of guanosin-8-yl-2-aminofluorene. • The transacetylase activity of rat liver is localized in the cytosol fraction (105000 × g supernatant) and is eluted at pH 7.4 shortly after the break-through peak upon chromatography on Sephadex G-75. The activity is maximal at pH 6.8 and is inhibited by thiol reagents. The activity is reduced to less than 10% by dialysis, and most of the activity can be restored by addition of pyridine nucleotides or cysteine. The increase in activity on addition of pyridine nucleotides to fresh undialyzed preparations is variable and usually does not exceed 20%. Of the compounds tested, aromatic hydroxamic acids were the only effective sources of acetyl groups; the highest yields of methylmercaptoaminoarenes were obtained with N-hydroxy -4-acetylaminobiphenyl or N-hydroxy-2-acetylaminofluorene as the acetyl donor. • This hepatic transacetylase activity was observed in rat, hamster and rabbit liver. Little or no activity was found with mouse, guinea pig, dog or human ( post-mortem) liver. The activity in rat liver decreased on administration of N-hydroxy-2-acetylaminofluorene; the enyzme activity was not affected by the oral administration of phenobarbital to rats. • The possible identity of this enzyme system with that studied by Booth ((1966) Biochem. J. 100, 745–753) and its possible role in the formation of bound 2-amino-fluorene derivatives on administration of N-hydroxy-2-acetylaminofluorene are discussed.