Acetyl transfer in arylamine metabolism

1. N-Hydroxyacetamidoaryl compounds (hydroxamic acids) are metabolites of arylamides, and an enzyme that transfers the acetyl group from these derivatives to arylamines has been found in rat tissues. The reaction products were identified by thin-layer chromatography and a spectrophotometric method, with 4-amino-azobenzene as acetyl acceptor, was used to measure enzyme activity. 2. The acetyltransferase was in the soluble fraction of rat liver, required a thiol for maximum activity and had a pH optimum between 6·0 and 7·5. 3. The soluble fractions of various rat tissues showed decreasing activity in the following order: liver, adrenal, kidney, lung, spleen, testis, heart; brain was inactive. 4. With the exception of aniline and aniline derivatives all the arylamines tested were effective as acetyl acceptors but aromatic compounds with side-chain amino groups were inactive. 5. The N-hydroxyacetamido derivatives of 2-naphthylamine, 4-amino-biphenyl and 2-aminofluorene were active acetyl donors but N-hydroxyacetanilide showed only slight activity. Acetyl-CoA was not a donor. 6. Some properties of the enzyme are compared with those of other acetyltransferases.     

Formation of N-acetylglutamate by extracts of higher plants

The enzymic synthesis of N-acetylglutamate was studied in extracts of higher plant tissues, especially in sugar beet leaves (Beta vulgaris L.). Sugar beet leaves had an enzyme that transferred the acetyl group either from acetyl-CoA or from N²-acetylornithine to glutamate. The enzyme was so unstable that special precautions were necessary for its detection and appreciable purification was impossible. The Km values were 2.5 and 0.025 mM for acetyl-CoA and N²-acetylornithine, respectively. The Km for glutamate was 23 mM with acetylornithine-glutamate transacetylase and 2.7 mM with acetyl-CoA-glutamate transacetylase. The pH optimum for acetyl-CoA-glutamate transacetylase was about 7.2 whereas that for acetylornithine-glutamate transacetylase was about 8.3. Acetylphosphate, N²-acetyl-2,4-diaminobutyrate, propionyl-CoA, and succinyl-CoA were not substrates.     

The occurrence of N-acetylaspartate amidohydrolase (aminoacylase II) in the developing rat

Of The amino acids and derivatives, N-acetyl-L-aspartic acid is present in a uniquely high level (5–6 μmol/g) in the brain of mammals after myelination has occurred. Much lower levels (0·06–0·17 μmol/g) are found prior to this stage of brain development (Tallan , 1957). In non-nervous tissues, on the other hand, only trace amounts of this acetyl amino acid are present (Tallan , Moore and Stein , 1956). N-acetyl aspartic acid serves as an excellent source of acetyl groups for lipogenesis in the developing rat brain (D'Adamo and Yatsu , 1966; Dadamo , Gidez and Yatsu , 1968). Non-nervous tissues such as kidney and mammary gland also rapidly metabolize the acetyl amino acid, the former tissue converting the acetyl group primarily to CO₂ and the latter to fatty acids (Benuck and D'Adamo , 1968). An enzyme with a high specificity for N-acetyl-L-aspartic acid initially termed aminoacylase II, was originally isolated from hog kidney by Birnbaum et al. (1952). Since the physiological role of the substrate is not known, it was of interest to study the occurrence of this enzyme, N-acetyl-L-aspartate amidohydrolase (EC 3.5.1.15), in developing tissues of the rat.     

Deacetylation of N8-acetylspermidine by subcellular fractions of rat tissue

The in vitro deacetylation of N⁸-acetylspermidine by an enzyme activity in rat tissues is described. This deacetylase activity occurs as a soluble, cytoplasmic enzyme in rat liver and was detected in the 100,000g supernatant fraction of all tissues examined. The highest specific activity was found in liver. Spleen, kidney, and lung were found to contain 20–50% of the activity in liver, while heart, brain, and skeletal muscle exhibited from 2 to 10% of the activity in liver. Serum contained only barely detectable levels of activity, much lower than any of the tissues studied. The in vitro metabolism of N¹-acetylspermidine differed from that observed for N⁸-acetylspermidine and does not appear to involve a simple deacetylation reaction.     

Purification and characterization from rat kidney membranes of a novel platelet-activating factor (PAF)-dependent transacetylase that catalyzes the hydrolysis of PAF, formation of PAF analogs, and C2-ceramide

We have previously identified two enzyme activities that transfer the acetyl group from platelet-activating factor (PAF) in a CoA-independent manner to lysoplasmalogen or sphingosine in HL-60 cells, endothelial cells, and a variety of rat tissues. These were termed as PAF:lysoplasmalogen (lysophospholipid) transacetylase and PAF:sphingosine transacetylase, respectively. In the present study, we have solubilized and purified this PAF-dependent transacetylase 13,700-fold from rat kidney membranes (mitochondrial plus microsomal membranes) based on the PAF:lysoplasmalogen transacetylase activity. The mitochondria and microsomes were prepared and washed three times, then solubilized with 0.04% Tween 20 at a detergent/protein (w/w) ratio of 0.1. The solubilized fractions from mitochondria and microsomes were combined and subjected to sequential column chromatographies on DEAE-Sepharose, hydroxyapatite, phenyl-Sepharose, and chromatofocusing. The enzyme was further purified by native-polyacrylamide gel electrophoresis (PAGE) and affinity gel matrix in which the competitive inhibitor of the enzyme, 1-O-hexadecyl-2-N-methylcarbamyl-sn-glycero-3-phosphoethanolamine was covalently attached to the CH-Sepharose. On SDS-PAGE, the purified enzyme showed a single homogeneous band with an apparent molecular mass of 40 kDa. The purified enzyme catalyzed transacetylation of the acetyl group not only from PAF to lysoplasmalogen forming plasmalogen analogs of PAF, but also to sphingosine producing N-acetylsphingosine (C2-ceramide). In addition, this enzyme acted as a PAF-acetylhydrolase in the absence of lipid acceptor molecules. These results suggest that PAF-dependent transacetylase is an enzyme that modifies the cellular functions of PAF through generation of other diverse lipid mediators.     

Properties and tissue distribution of a novel aldo-keto reductase encoding in a rat gene (Akr1b10)

A recent rat genomic sequencing predicts a gene Akr1b10 that encodes a protein with 83% sequence similarity to human aldo-keto reductase (AKR) 1B10. In this study, we isolated the cDNA for the rat AKR1B10 (R1B10) from rat brain, and examined the enzymatic properties of the recombinant protein. R1B10 utilized NADPH as the preferable coenzyme, and reduced various aldehydes (including cytotoxic 4-hydroxy-2-hexenal and 4-hydroxy- and 4-oxo-2-nonenals) and α-dicarbonyl compounds (such as methylglyoxal and 3-deoxyglucosone), showing low K_m values of 0.8-6.1 μM and 3.7-67 μM, respectively. The enzyme also reduced glyceraldehyde and tetroses (K_m = 96-390 μM), although hexoses and pentoses were inactive and poor substrates, respectively. Among the substrates, 4-oxo-2-nonenal was most efficiently reduced into 4-oxo-2-nonenol, and its cytotoxicity against bovine endothelial cells was decreased by the overexpression of R1B10. R1B10 showed low sensitivity to aldose reductase inhibitors, and was activated to approximately two folds by valproic acid, and alicyclic and aromatic carboxylic acids. The mRNA for R1B10 was expressed highly in rat brain and heart, and at low levels in other rat tissues and skin fibroblasts. The results suggest that R1B10 functions as a defense system against oxidative stress and glycation in rat tissues.     

Platelet-activating factor acetylhydrolase and transacetylase activities in human aorta and mammary artery

Platelet-activating factor (PAF), the potent phospholipid mediator of inflammation, is involved in atherosclerosis. Platelet-activating factor-acetylhydrolase (PAF-AH), the enzyme that inactivates PAF bioactivity, possesses both acetylhydrolase and transacetylase activities. In the present study, we measured acetylhydrolase and transacetylase activities in human atherogenic aorta and nonatherogenic mammary arteries. Immunohistochemistry analysis showed PAF-AH expression in the intima and the media of the aorta and in the media of mammary arteries. Acetylhydrolase and transacetylase activities were (mean +/- SE, n = 38): acetylhydrolase of aorta, 2.8 +/- 0.5 pmol/min/mg of tissue; transacetylase of aorta, 3.3 +/- 0.7 pmol/min/mg of tissue; acetylhydrolase of mammary artery, 1.4 +/- 0.3 pmol/min/mg of tissue (P < 0.004 as compared with acetylhydrolase of aorta); transacetylase of mammary artery, 0.8 +/- 0.2 pmol/min/mg of tissue (P < 0.03 as compared with acetylhydrolase of mammary artery). Lyso-PAF accumulation and an increase in PAF bioactivity were observed in the aorta of some patients. Reverse-phase HPLC and electrospray ionization mass spectrometry analysis revealed that 1-O-hexadecyl-2 acetyl-sn glycero-3-phosphocholine accounted for 60% of the PAF bioactivity and 1-O-hexadecyl-2-butanoyl-sn-glycerol-3-phosphocholine for 40% of the PAF bioactivity. The nonatherogenic properties of mammary arteries may in part be due to low PAF formation regulated by PAF-AH activity. In atherogenic aortas, an imbalance between PAF-AH and transacetylase activity, as well as lyso-PAF accumulation, may lead to unregulated PAF formation and to progression of atherosclerosis.     

Deacylation of carcinogenic 5-nitrofuran derivatives by mammalian tissues

The deacylations of N-[4-(5-nitro-2-furyl)-2-thiazolyl] fonnamide (FANFT), N-[4-(5-nitro-2-furyl)-2-thiazolyl] acetarnide (NFTA) and formic acid 2-[4-(5-nitro-2-furyl)-2-thiazolyl] hydrazide (FNT) by liver, kidney, small intestines and stomach of mouse, rat, hamster and guinea pig were investigated. FANFT was deformylated to 2-amino-4-(5-nitro-2-furyl)thiazole (ANFT). FANFT formamidase activity was higher in the liver and small intestines of mouse, hamster and guinea pig, and small intestines and stomach of rat. There was no detectable FANFT formamidase activity in the stomach of the mouse and hamster. Neither NFTA nor FNT was deacylated by the rodent tissue homogenates studied. It is suggested that (1)4 ANFT is a metabolite of FANFT but not NFTA; (2) 2-hydrazino-4-(5-nitro-2-furyl)thiazole (HNFT) may not be a metabolite of FNT; and (3) the induction of tumors by FANFT, NFTA and FNT may not be due to a common carcinogenic metabolite, although these chemicals demonstrate similar organ specificities in some of these rodents.     

Role of maternal tissue in the synthesis of polyunsaturated fatty acids in response to a lipid-deficient diet during pregnancy and lactation in rats

During pregnancy and lactation, metabolic adaptations involve changes in expression of desaturases and elongases (Elovl2 and Elovl5) in the mammary gland and liver for the synthesis of long-chain polyunsaturated fatty acids (LC-PUFAs) such as arachidonic acid (AA) required for fetal and postnatal growth. Adipose tissue is a pool of LC-PUFAs. The response of adipose tissue for the synthesis of these fatty acids in a lipid-deficient diet of dams is unknown. The aim of this study was to explore the role of maternal tissue in the synthesis of LC-PUFAs in rats fed a low-lipid diet during pregnancy and lactation. Fatty acid composition (indicative of enzymatic activity) and gene expression of encoding enzymes for fatty acid synthesis were measured in liver, mammary gland and adipose tissue in rats fed a low-lipid diet. Gene expression of desaturases, elongases, fatty acid synthase (Fasn) and their regulator Srebf-1c was increased in the mammary gland, liver and adipose tissue of rats fed a low-lipid diet compared with rats from the adequate-lipid diet group throughout pregnancy and lactation. Genes with the highest (P < 0.05) expression in the mammary gland, liver and adipose tissue were Elovl5 (1333%), Fads2 (490%) and Fasn (6608%), respectively, in a low-lipid diet than in adequate-lipid diet. The percentage of AA in the mammary gland was similar between the low-lipid diet and adequate-lipid diet groups during the second stage of pregnancy and during lactation. The percentage of monounsaturated and saturated fatty acids was significantly (P < 0.05) increased throughout pregnancy and lactation in all tissues in rats fed a low-lipid diet than in rats fed an adequate-lipid diet. Results suggest that maternal metabolic adaptations used to compensate for lipid-deficient diet during pregnancy and lactation include increased expression of genes involved in LC-PUFAs synthesis in a stage- and tissue-specific manner and elevated lipogenic activity (saturated and monounsaturated fatty acid synthesis) of maternal tissues including adipose tissue.     

Partial characterization of N-acetyltransferase activity from cerebral ganglia and malpighian tubules of Periplaneta americana

Octopamine-N-acetyltransferase (NAT) activity from Malpighian tubules and cerebral ganglia of Periplaneta americana was studied using high performance liquid chromatography with coulometric electrochemical detection. The enzyme from these tissues is highly soluble and temperature-resistant with maximal activity demonstrated at 50°C. The pH profiles of enzyme activity from Malpighian tubules and cerebral ganglia differ markedly although, in both tissues, strong activity is evident under alkaline conditions. Kinetic analyses indicate that the enzyme present in Malpighian tubules has a K_m for octopamine of 0.40 mM with a maximum velocity of 37.9 nmol N-acetyl-p-octopamine (N-acOA) produced/mg protein/min; the equivalent values for acetyl CoA were determined as 0.32 mM and 36.9 nmol N-acOA/mg protein/min. NAT from cerebral ganglia showed K_ms for octopamine and acetyl CoA of 0.33 and 0.19 mM, respectively, and the maximum velocity with octopamine was 6.8 nmol N-acOA/mg protein/min. Analysis of NAT activity from a variety of cockroach tissues indicates that the enzyme is widely distributed, with the gut and associated Malpighian tubules, conglobate gland and salivary glands showing the greatest activity.     

Identity of β-alanine-oxo-glutarate aminotransferase and L-β-aminoisobutyrate aminotransferase in rat liver

L-β-Aminoisobutyrate served as an amino donor for purified β-alanine-oxo-glutarate aminotransferase from rat liver when 2-oxoglutarate was employed as an amino acceptor, but he D-isomer did not. L-β-Aminoisobutyrate acted as a competitive inhibitor with respect to β-alanine and had a K_i of approximately 2.6 mM, which is the same value as the K_m of 2.7 mM. When the crude extract was applied to a DEAE-Sepharose CL-6B column, L-β-aminoisobutyrate aminotransferase and β-alanine-oxo-glutarate aminotransferase activities were found in the same fractions with a single peak. Antiserum to rat liver β-alanine-oxo-glutarate aminotransferase inhibited L-β-aminoisobutyrate aminotransferase activity in rat liver in the same way as β-alanine-oxo-glutarate aminotransferase activity.     

The coenzyme A requirement of mammalian fatty acid synthetase: evidence for involvement in the elongation of acyl-enzyme thioesters

We have confirmed that coenzyme A is required for rat fatty acid synthetase activity (T. C. Linn, M. J. Stark, and P. A. Srere, 1980, J. Biol. Chem.255, 1388–1392). When rat liver or mammary gland fatty acid synthetase was assayed in the presence of a CoA-scavenging system such as ATP citrate lyase, almost complete inhibition of fatty acid synthesis was observed. The inhibition was reversed by addition of CoA or pantetheine, but not by addition of N-acetylcysteamine or other thiols. In the absence of CoA, the rate of elongation of acyl moieties on both native fatty acid synthetase and fatty acid synthetase lacking the chain-terminating thioesterase I component (trypsinized fatty acid synthetase) was reduced 100-fold. All of the palmitate synthesized slowly by the CoA-depleted native multienzyme was released, by the thioesterase I component, as the free fatty acid; only shorter-chainlength acyl moieties remained bound to the enzyme. The acyl-S-multienzyme thioesters formed by the trypsinized fatty acid synthetase in the absence of CoA contained saturated moieties of chain length C₆-C₁₆; addition of CoA promoted elongation of the acyl-S-multienzyme thioesters without release from the enzyme. The transfer of acetyl and malonyl moieties from CoA to the multienzyme, the reduction of S-acetoacetyl-N-acetylcysteamine and S-crotonyl-N-acetylcysteamine, and the dehydration of S-β-hydroxybutyryl-N-acetylcysteamine, reactions catalyzed by the fatty acid synthetase, were not dependent on the presence of CoA. The hydrolysis of acyl-S-multienzyme catalyzed by thioesterase I, the resident chain-terminating component of the fatty acid synthetase, and thioesterase II, a monofunctional mammary gland chain-terminating enzyme, was also independent of CoA availability as was hydrolysis of an acyl-S-pantetheine pentapeptide isolated from the multienzyme. On the basis of these observations we conclude that CoA is required for the elongation of acyl moieties on the fatty acid synthetase but not for their release from the multienzyme.     

Proteomic analysis of endogenous conjugated linoleic acid biosynthesis in lactating rats and mouse mammary gland epithelia cells (HC11)

This study was conducted to investigate the amount of CLA synthesized endogenously by rat mammary tissues in response to TVA (a precursor for cis-9, trans-11 CLA endogenous synthesis) treatment as well as the differences in the protein expression of genes encoding the biosynthesis of CLA in rat mammary tissue and mouse mammary gland epithelia cells (HC11). Treatment with TVA resulted in improved CLA productivity. Furthermore, 2-DE revealed two spots in samples of mammary tissues and one spot in samples of mammary gland epithelia cells (HC11) that were consistently altered in the TVA treatment groups when compared with the control group (non-fatty acid). The mRNA expression patterns of three of the proteins (PDI, PRDX2, LAMR1), as measured by real-time PCR, were similar to the pattern of protein abundance. In addition, the expression of SCD mRNA in the mammary tissue of rats and HC11 cell treated with TVA was higher than in the control group. Our results suggest that the identified proteins may be related to CLA biosynthesis in mammary tissue.     

Metabolism of Monoterpenes: Acetylation of (-)-Menthol by a Soluble Enzyme Preparation from Peppermint (Mentha piperita) Leaves

The essential oil from mature leaves of flowering peppermint (Mentha piperita L.) contains up to 15% (—)-menthyl acetate, and leaf discs converted exogenous (—)-[G-³H]menthol into this ester in approximately 15% yield of the incorporated precursor. Leaf extracts catalyzed the acetyl coenzyme A-dependent acetylation of (—)-[G-³H]menthol and the product of this transacetylase reaction was identified by radiochromatographic techniques. Transacetylase activity was located mainly in the 100,000g supernatant fraction, and the preparation was partially purified by combination of Sephadex G-100 gel filtration and chromatography on O-diethylaminoethyl-cellulose. The transacetylase had a molecular weight of about 37,000 as judged by Sephadex G-150 gel filtration, and a pH optimum near 9. The apparent K_m and velocity for (—)-menthol were 0.3 mm and 16 nmol/hr· mg of protein, respectively. The saturation curve for acetyl coenzyme A was sigmoidal, showing apparent saturation near 0.1 mm. Dithioerythritol was required for maximum activity and stability of the enzyme, and the enzyme was inhibited by thiol directed reagents such as p-hydroxymercuribenzoate. Diisopropylfluorophosphate also inhibited transacylation suggesting the involvement of a serine residue in catalysis. The transacylase was highly specific for acetyl coenzyme A; propionyl coenzyme A and butyryl coenzyme A were not nearly as efficient as acyl donors (11% and 2%, respectively). However, the enzyme was much less selective with regard to the alcohol substrate, suggesting that the nature of the acetate ester synthesized in mint is more dependent on the type of alcohol available than on the specificity of the transacetylase. This is the first report on an enzyme involved in monoterpenol acetylation in plants. A very similar enzyme, catalyzing this key reaction in the metabolism of menthol, was also isolated from the flowers of peppermint.     

Further studies on a glycolipid formed from dolichyl-D-glucosyl monophosphate

Incubation of liver microsomes with dolichyl-d-glucosyl-¹⁴C monophosphate led to the labelling of an endogenous acceptor. This compound seems to be also a dolichol derivative. It contains a high-molecular weight oligosaccharide bound to dolichol through a phosphate or pyrophosphate bond. Various treatments of the labelled oligosaccharide afforded further information on its structure: Reduction with sodium borohydride, followed by acid hydrolysis gave only radioactive d-glucose indicating that the labelled d-glucose is not incorporated at the reducing end of the oligosaccharide. The percentage of radioactivity, liberated as formic acid after periodate oxidation, indicates that more than one molecule of d-glucose is incorporated and that at least one of them is inside the oligosaccharide chain. Alkaline treatment of the otherwise neutral oligosaccharide gave two positively charged derivatives which could be neutralized by N-acetylation, indicating the presence of two hexosamine residues. The oligosaccharides isolated from different tissues by the same method as that used for rat liver, were similar as judged by their migration in paper chromatography and by the pattern of products liberated by acetolysis.     

Glycosyltransferases with endogenous acceptor activity in plasma membranes isolated from rat liver

Plasma membrane fractions from rat liver exhibited glycosyltransferase activity with endogenous membrane-associated acceptors and either UDP-galactose, UDPglucose, UDP-N-acetylglucosamine, or GDPmannose donors. Of these, incorporation into non-lipid acceptors was most active with UDP-galactose and only with UDPgalactose and UDPmannose was there incorporation into endogenous lipid acceptors. CMP-N-acetylneuraminic acid was inactive as a donor with the isolated plasma membranes. In order to demonstrate transferase activity, low concentrations of substrate sugar nucleotides and short incubation times were used as well as sulfhydryl protectants and a phosphatase inhibitor (NaF) in the reaction mixtures. The findings support the concept of surface localization of at least a galactosyl transferase in cells of rat liver.     

Synthesis and biological evaluation of 2-quinolineacrylamides

A series of C6-substituted N-hydroxy-2-quinolineacrylamides (3–15), with four types of bridging groups have been synthesized. Most of these compounds exhibit antiproliferative activity against A549 and HCT116 cells and Western blot analysis revealed that they are able to inhibit HDAC. Measurement of the HDAC isoform activity of ether-containing compounds showed that compound 9 has distinct HDAC6 selectivity, more than 300-fold over other isoforms. This paper describes the development of 6-aryloxy-N-hydroxy-2-quinolineacrylamides as potential HDAC6 inhibitors.     

Synthesis,crystal and antibacterial studies of diversely functionalized tetrahydropyridin-4-ol

In an effort to expand the spectrum of antibacterial activity associated with piperidin-4-one derivatives, we have synthesized two series of 3-carboxyethyl-2,6-diphenyl-4-hydroxy-Δ³-tetrahydropyridine derivatives bearing diversified heterocyclic and aromatic systems at the nitrogen atom through acetyl (6–18) and 2-propanoyl (9–31) linkers. Unlike acetyl derivatives, NMR spectral pattern of the propanoyl counterparts revealed the existence of pair of rotational isomers (syn and anti) in solution at room temperature due to the hindered rotation about N–CO bond. X-ray crystal studies of 9 and 24 clearly pointed out that all the compounds existed in only one form particularly, in stable syn form in solid state. Each of the compounds was screened for their in vitro antibacterial activity against nine human pathogenic Gram-positive strains including multiple drug resistant organisms and seven problematic Gram-negative strains. Among the various heterocycles examined here, imidazole substituted derivatives 12 and 25 exhibited antibacterial activity approaching that of Linezolid and Trovafloxacin drugs particularly against multiple resistant Enterococcus faecium-VanA phenotype strains.     

Immunochemical studies with soluble and mitochondrial pyruvate carboxylase activities from rat tissues

1. Pyruvate carboxylase (EC 6.4.1.1), purified from rat liver mitochondria to a specific activity of 14 units/mg, was used for the preparation of antibodies in rabbits. 2. Tissue distribution studies showed that pyruvate carboxylase was present in all rat tissues that were tested, with considerable activities both in gluconeogenic tissues such as liver and kidney and in tissues with high rates of lipogenesis such as white adipose tissue, brown adipose tissue, adrenal gland and lactating mammary gland. 3. Immunochemical titration experiments with the specific antibodies showed no differences between the inactivation of pyruvate carboxylase from mitochondrial or soluble fractions of liver, kidney, mammary gland, brown adipose tissue or white adipose tissue. 4. The antibodies were relatively less effective in reactions against pyruvate carboxylase from sheep liver than against the enzyme from rat tissues. 5. Pyruvate carboxylase antibodies did not inactivate either propionyl-CoA carboxylase or acetyl-CoA carboxylase from rat liver. 6. It is concluded that pyruvate carboxylase in lipogenic tissues is similar antigenically to the enzyme in gluconeogenic tissues and that the soluble activities of pyruvate carboxylase detected in many rat tissues do not represent discrete enzymes but are the result of mitochondrial damage during tissue homogenization.     

Glucose-6-phosphate as a probe for the glucosamine-6-phosphate N-acetyltransferase Michaelis complex

Glucosamine-6-phosphate N-acetyltransferase (GNA1) catalyses the N-acetylation of d-glucosamine-6-phosphate (GlcN-6P), using acetyl-CoA as an acetyl donor. The product GlcNAc-6P is an intermediate in the biosynthesis UDP-GlcNAc. GNA1 is part of the GCN5-related acetyl transferase family (GNATs), which employ a wide range of acceptor substrates. GNA1 has been genetically validated as an antifungal drug target. Detailed knowledge of the Michaelis complex and trajectory towards the transition state would facilitate rational design of inhibitors of GNA1 and other GNAT enzymes. Using the pseudo-substrate glucose-6-phosphate (Glc-6P) as a probe with GNA1 crystals, we have trapped the first GNAT (pseudo-)Michaelis complex, providing direct evidence for the nucleophilic attack of the substrate amine, and giving insight into the protonation of the thiolate leaving group.