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PubMed1988

Acetyl-coenzyme A:polysialic acid O-acetyltransferase from K1-positive Escherichia coli. The enzyme responsible for the O-acetyl plus phenotype and for O-acetyl form variation.

The capsular polysaccharide of Escherichia coli K1 is a linear polymer of N-acetylneuraminic acid in alpha-2,8 linkage. Certain substrains of E. coli K1 (designated OAc+) modify the polysaccharide by O-acetylation of the sialic acids. We demonstrate here an acetyl-coenzyme A: polysialosyl O-acetyltransferase activity that is found only in E. coli K1 OAc+ substrains. When form variation between the O-acetyl-positive and -negative states occurred in strain D698:K1, the fluctuations were accompanied by appropriate changes in the expression of enzyme activity. Thus, expression of this enzyme can account for the OAc+ phenotype and for the form variation between OAc+ and OAc-. The enzyme was solubilized in nonionic detergent and freed of endogenous acceptor activity by DEAE-cellulose chromatography, and its general properties were determined. Analysis of the reaction product showed a highly preferential acetylation reaction that was confined to polysialosyl units of greater than 14 residues. Acetyl groups were shown to be transferred to both the 7- and the 9-positions of the sialic acid residues. The partially purified enzyme was stable even after prolonged incubation at 57 degrees C. In contrast, any further purification resulted in loss of activity, even at 4 degrees C. Treatment of the stable enzyme with a polysialic acid-specific endoneuraminidase caused a similar loss of enzyme stability. This effect of the endoneuraminidase could be protected against by the addition of exogenous polysialic acid. This indicates that the partially purified enzyme contains traces of endogenous polysialic acid substrate that are required for the stability of the enzyme. Finally, the enzyme can O-acetylate the polysialic acid chains on the eucaryotic protein neural cell adhesion molecule, suggesting that enzymatic recognition of the substrate requires only the polysialic acid sequence.

Acetylation↗
PubMed2002

Reactivity and selectivity of the N-acetyl-Glu-P-1, N-acetyl-Glu-P-2, N-acetyl-MeIQx, and N-acetyl-IQx nitrenium ions: comparison to carbocyclic N-arylnitrenium ions.

The model ultimate carcinogens 1a-d, related to the metabolites of the food-derived carcinogenic heterocyclic amines Glu-P-1, Glu-P-2, MeIQx, and IQx, spontaneously decompose in neutral aqueous solution to generate the heterocyclic nitrenium ions, 2a-d. The less reactive esters 1a and 1b also undergo acid-catalyzed ester hydrolysis to generate the corresponding hydroxamic acids at pH <2, while the more reactive 2c and 2d are prone to rearrangement in nonaqueous solvents. The reactions of the nitrenium ions with AcO(-), HPO(4)(2-), N(3)(-), and 2'-deoxyguanosine (d-G) were characterized in aqueous solution by using a combination of competitive trapping methods and product isolation and identification. The reactions with N(3)(-) and d-G generally follow patterns previously established for carbocyclic nitrenium ions, but the reactions with AcO(-) and HPO(4)(2-) are unusual. Similar reactions have previously only been reported for heterocyclic 1-alkyl-2-imidazolium ions. The N(3)(-)/solvent selectivities of these ions (5.1 x 10(6) M(-1) for 2a, 2.3 x 10(6) M(-1) for 2b, 1.2 x 10(5) M(-1) for 2c, and 5.2 x 10(4) M(-1) for 2d) are comparable to those of highly selective carbocyclic nitrenium ions. If k(az) for these ions is diffusion limited at ca. 5 x 10(9) M(-1) s(-1) the aqueous solution lifetimes of these ions range from 10 micros (2d) to 1 ms (2a). These ions are also highly selective for trapping by d-G, but comparisons to other nitrenium ions show that they are 10- to 50-fold less selective for trapping by d-G than they would be if both the N(3)(-) and d-G reactions were diffusion limited. This is not a consequence of their heterocyclic structures. Several carbocyclic ions show similar behavior. The relatively inefficient trapping of 2c and 2d by d-G may account for the observation of the unusual minor N-2 d-G adduct that is isolated for both of these nitrenium ions, but has not previously been observed for the reactions of other nitrenium ions with monomeric d-G.

Acetylation↗
PubMed1989

3,4-Dinitrophenyl N-acetyl-beta-D-glucosaminide, a synthetic substrate for direct spectrophotometric assay of N-acetyl-beta-D-glucosaminidase or N-acetyl-beta-D-hexosaminidase.

3,4-Dinitrophenyl N-acetyl-beta-D-glucosaminide (3,4-dnpGlcNAc) was synthesized and proposed as an artificial substrate for N-acetyl-beta-D-glucosaminidase (EC 3.2.1.30) and N-acetyl-beta-D-hexosaminidase (EC 3.2.1.52) (collectively abbreviated as NAGase). 3,4-dnpGlcNAc is water soluble and is fairly stable in aqueous medium. 3,4-Dinitrophenol released from 3,4-dnpGlcNAc by NAGase absorbs light of 400 nm at pH near 5, where the activity of urinary NAGase is assayed for diagnostic use. Direct and sensitive spectrophotometric assay of NAGase is, thus, possible using 3,4-dnpGlcNAc as an artificial substrate by monitoring the increase of A400 due to the release of 3,4-dinitrophenol in a thermostated spectrophotometer.

Acetylglucosamine↗
PubMed2001

Isolation and structural analyses of positional isomers of 6(1),6(n)-di-O-(N-acetyl-beta-D-glucosaminyl)cyclomaltoheptaose (n=2, 3, and 4) and 6-O-[6-O-(N-acetyl-beta-D-glucosaminyl)-N-acetyl-beta-D-glucosaminyl]cyclomaltoheptaose.

6-O-[6-O-(N-acetyl-beta-D-glucosaminyl)-N-acetyl-beta-D-glucosaminyl]cyclomaltoheptaose (beta CD) and three positional isomers of 6(1),6(n)-di-O-(N-acetyl-beta-D-glucosaminyl)cyclomaltoheptaose (n=2, 3, and 4) in a mixture of products from beta CD and N-acetylglucosamine by the reversed reaction of beta-N-acetylhexosaminidase from jack bean were isolated and purified by HPLC. The structures of four isomers of di-N-acetylglucosaminyl-beta CDs were determined by FABMS and NMR spectroscopy. The degree of polymerization of the branched oligosaccharides produced by enzymatic degradation with bacterial saccharifying alpha-amylase (BSA) was established by LC-MS methods.

Acetylglucosamine↗
PubMed2001

Evaluation of De-O-acetylated meningococcal C polysaccharide-tetanus toxoid conjugate vaccine in infancy: reactogenicity, immunogenicity, immunologic priming, and bactericidal activity against O-acetylated and De-O-acetylated serogroup C strains.

The polysaccharide capsule of serogroup C Neisseria meningitidis (MenC) has been integral to vaccine development. Licensed MenC vaccines contain the O-acetylated (OAc+) form of polysaccharide. Some MenC strains have de-O-acetylated (OAc-) polysaccharide, which may affect antibody specificity and functional activity when used in a vaccine. We evaluated an OAc-MenC conjugate-tetanus toxoid conjugate (MCC-TT) vaccine given concomitantly with whole-cell diphtheria-tetanus-pertussis, Haemophilus influenzae type b, and oral polio immunization in 83 infants at 2, 3, and 4 months of age. Serum bactericidal activities (SBA) against OAc+ and OAc- MenC strains and OAc+ and OAc- polysaccharide-specific immunoglobulin G (IgG) levels were evaluated. MCC-TT vaccine was well tolerated. All infants produced SBA titers of > or = 8 after a single dose at 2 months of age. The SBA geometric mean titer for OAc+ strain C11 increased from 2.7 (95% confidence interval [CI] 2.2 to 3.2) to 320 (95% CI, 237 to 432), 773 (95% CI, 609 to 982), and 1,063 (95% CI, 856 to 1319) after one, two, and three doses of MCC-TT, respectively. OAc- IgG levels were twice as high as OAc+ IgG levels after the primary series of MCC-TT vaccine, and the SBA was significantly higher against the OAc- MenC strain. Antibody responses to booster vaccination with either OAc+ MenC polysaccharide vaccine (MACP) or a fourth dose of MCC-TT at 14 months of age provided evidence of immunologic memory. The acetylation status of the booster vaccine influenced the specificity of the response, with significantly higher OAc- IgG levels and SBA after MCC-TT vaccine compared to MACP vaccine but similar OAc+ antibody levels. MCC-TT vaccine is highly immunogenic and primes for immunologic memory against OAc+ and OAc- MenC strains in infancy.

Antibodies, Bacterial↗
PubMed1988

Synthesis and mass spectra of 4-O-acetyl-1,5-anhydro-2,3,6-tri-O-ethyl-D-glucitol and the positional isomers of 4-O-acetyl-1,5-anhydro-di-O-ethyl-O- methyl-D-glucitol and 4-O-acetyl-1,5-anhydro-O-ethyl-di-O-methyl-D-glucitol.

Reductive cleavage of fully methylated, partially O-ethylated cellulose or fully ethylated, partially O-methylated cellulose and subsequent acetylation had previously been shown to produce 4-O-acetyl-1,5-anhydro-2,3,6-tri-O-methyl-, -6- O-ethyl-2,3-di-O-methyl-, -3-O-ethyl-2,6-di-O-methyl-, -2-O-ethyl-3,6-di-O- methyl-, -2,3-di-O-ethyl-6-O-methyl-, -2,6-di-O-ethyl-3-O-methyl-, -3,6-di-O- ethyl-2-O-methyl-, and -2,3,6-tri-O-ethyl-D-glucitol. Described herein is the independent synthesis of these derivatives, except for the first (which had been reported); and their 1H-n.m.r. spectra, chemical-ionization (NH3) mass spectra, and electron-impact mass spectra are tabulated.

Chemical Phenomena↗
PubMed2005

Comparative structural analysis of 5,6,7,9-tetra-O-acetyl-4,8-anhydro-1,3-dideoxy-D-glycero-L-gluco-nonulose and its 1-O-acetylated analog, 1,2,3,4,6-penta-O-acetyl-beta-D-galactopyranose using X-ray crystallography.

Comparative X-ray diffraction analysis of 5,6,7,9-tetra-O-acetyl-4,8-anhydro-1,3-dideoxy-D-glycero-L-gluco-nonulose (1) and a structurally related analog, 1,2,3,4,6-penta-O-acetyl-beta-D-galactopyranose (2), are reported. Both crystals have one molecule in the unit cell and the pyranose rings in both exist in the 4C1 conformation.

Crystallization↗
PubMedDate not supplied

Kinetic characterization of acetylator genotype-dependent and -independent N-acetyltransferase isozymes in homozygous rapid and slow acetylator inbred hamster liver cytosol.

Acetyl-coenzyme A (AcCoA)-dependent arylamine N-acetyltransferase (NAT) activity (EC 2.3.1.5) was examined in liver cytosol derived from homozygous rapid acetylator (Bio. 87.20) and homozygous slow acetylator (Bio. 82.73/H) Syrian inbred hamsters. Expression of NAT activity toward p-aminobenzoic acid (PABA), 2-aminofluorene (AF), and 4-aminobiphenyl (ABP) was acetylator genotype-dependent, whereas N-acetyltransferase activity toward isoniazid was acetylator genotype-independent. Two isozymes of NAT activity were partially purified by anion exchange fast protein liquid chromatography from the hepatic cytosol of both homozygous rapid and homozygous slow acetylator hamsters. The first eluting NAT isozyme exhibited a polymorphic expression toward AF, ABP, and PABA although the second eluting NAT isozyme exhibited a monomorphic expression across acetylator genotypes toward the same substrates. Determination of Michaelis-Menten kinetic constants in hepatic cytosol of homozygous rapid and slow acetylator hamsters suggests that PABA, AF, and ABP NAT activities were acetylator genotype-dependent because of catalysis by polymorphic NAT isozyme that is both an apparent Km and Vmax variant, whereas, the acetylator genotype-independent expression of isoniazid NAT activity appeared to result from catalysis via a common monomorphic NAT isozyme in both acetylator genotypes. Additional kinetic studies on the partially purified NAT isozymes of homozygous rapid and slow acetylator hamster liver confirmed that the polymorphic NAT isozyme exhibited a substantially higher apparent maximum velocity in homozygous rapid acetylators than slow acetylators toward PABA, AF, and ABP as well as acetylator genotype-related differences in the apparent Km toward each of these substrates. In contrast, the monomorphic NAT isozyme of both acetylator genotypes showed apparent Vmax levels of NAT activity that did not vary with acetylator genotype. Furthermore, the monomorphic NAT isozyme did not show acetylator genotype-related variations in apparent Km toward the arylamine carcinogens AF and ABP, although differences were noted for PABA and AcCoA. These results suggest that the acetylator genotype-dependent expression of AcCoA-dependent NAT activity in hamster hepatic cytosol toward arylamines is primarily accountable by structural variants (allozymes) of polymorphic NAT under the genetic regulation of the acetylator gene locus. The acetylator genotype-independent expression of isoniazid NAT activity is attributable to a common monomorphic NAT isozyme in both acetylator genotypes.

4-Aminobenzoic Acid↗
PubMed1990

Biosynthesis and turnover of O-acetyl and N-acetyl groups in the gangliosides of human melanoma cells.

We and others previously described the melanoma-associated oncofetal glycosphingolipid antigen 9-O-acetyl-GD3, a disialoganglioside O-acetylated at the 9-position of the outer sialic acid residue. We have now developed methods to examine the biosynthesis and turnover of disialogangliosides in cultured melanoma cells and in Golgi-enriched vesicles from these cells. O-Acetylation was selectively expressed on di- and trisialogangliosides, but not on monosialogangliosides, nor on glycoprotein-bound sialic acids. Double-labeling of cells with [3H]acetate and [14C]glucosamine introduced easily detectable labels into each of the components of the ganglioside molecules. Pulse-chase studies of such doubly labeled molecules indicated that the O-acetyl groups turn over faster than the parent molecule. When Golgi-enriched vesicles from these cells were incubated with [acetyl-3H]acetyl-coenzyme A, the major labeled products were disialogangliosides. [Acetyl-3H]O-acetyl groups were found at both the 7- and the 9-positions, indicating that both 7-O-acetyl GD3 and 9-O-acetyl GD3 were synthesized by the action of O-acetyltransferase(s) on endogenous GD3. Analysis of the metabolically labeled molecules confirmed the existence of both 7- and 9-O-acetylated GD3 in the intact cells. Surprisingly, the major 3H-labeled product of the in vitro labeling reaction was not O-acetyl-GD3, but GD3, with the label exclusively in the sialic acid residues. Fragmentation of the labeled sialic acids by enzymatic and chemical methods showed that the 3H-label was exclusively in [3H]N-acetyl groups. Analyses of the double-labeled sialic acids from intact cells also showed that the 3H-label from [3H]acetate was exclusively in the form of [3H]N-acetyl groups, whereas the 14C-label was at the 4-position. Pulse-chase analysis of the 3H/14C ratio showed that the N-acetyl groups of both GD3 and of the monosialoganglioside GM3 were turning over faster than the parent molecules. Selective periodate oxidation showed that both the inner and outer sialic acid residues of GD3 incorporated 3H-label in the in vitro reaction, and showed similar turnover of N-acetylation in the pulse-chase study. Taken together, these results indicate that both the O- and N-acetyl groups of the sialic acid residues of gangliosides turn over faster than the parent molecules. They also demonstrate a novel re-N-acetylation reaction that predicts the existence of de-N-acetyl gangliosides in melanoma cells.

Acetates↗
PubMed2004

Acetylation of the chemotaxis response regulator CheY by acetyl-CoA synthetase purified from Escherichia coli.

Acetylation of CheY, the excitatory response regulator of bacterial chemotaxis, by the enzyme acetyl-CoA synthetase (Acs) is involved in Escherichia coli chemotaxis, but its function is obscure. Here, we overproduced Acs from E.coli, purified it in quantities sufficient for biochemical work, and characterized both the enzyme and the CheY acetylation reaction that it catalyzes. Such characterization is essential for revealing the function of CheY acetylation in chemotaxis. The enzyme exhibited characteristics typical of prokaryotic Acs enzymes, and it could use either acetate or AcCoA as an acetyl donor for CheY acetylation. The Acs-catalyzed acetylation of CheY was reversible, an essential property for a regulatory process, and cooperative (Hill coefficient approximately 3). By Western blotting with specific anti-acetyl-lysine antibody we demonstrated that Acs undergoes autoacetylation, that CheY is acetylated to a small extent when isolated, and that the extent is elevated following in vitro acetylation. Exposing the intact protein to matrix-assisted laser desorption ionization time-of-flight mass spectrometry and electro-spray mass spectrometry, we found that, in most cases, purified CheY is a mixture of species having zero to six acetyl groups per molecule, with non-acetylated CheY being the most abundant species. By proteolytic in-gel digestion of non-treated CheY followed by peptide fingerprinting, precursor ion scan, and tandem mass spectrometry, we found that the acetylation sites of CheY are clustered at the C terminus of the protein, with lysine residues 91, 92, 109, 119, 122 and 126 being the main acetylation sites. Following in vitro acetylation, the main change that seemed to occur was an incremental increase in the extent of acetylation of the same lysine residues. Thus, CheY is similar to many eukaryotic proteins involved in signaling, which undergo both phosphorylation and multiple acetylation, and in which the acetylation sites are restricted to a particular region.

Acetylation↗
PubMed1990

Biochemical and serological characteristics of natural 9-O-acetyl GD3 from human melanoma and bovine buttermilk and chemically O-acetylated GD3.

Because its expression appears to be largely restricted to human melanomas, 9-O-acetyl-GD3 is a candidate antigen for vaccine construction. Searching for potential sources, we compared chemically O-acetylated calf brain GD3 and 9-O-acetyl-GD3 extracted from bovine buttermilk with 9-O-acetyl-GD3 from human melanoma. Three fractions (F1-F3) of chemically O-acetylated GD3 differed in the number and position of O-acetyl groups. O-Acetylation sites were the lactose portion in F1 and lactose as well as sialic acid in F2 and F3. Natural (melanoma- or buttermilk-derived) 9-O-acetyl-GD3 was O-acetylated solely on the sialic acid moiety. While F1 was not reactive with monoclonal antibodies against 9-O-acetyl-GD3, F2 and F3 were as reactive as the natural products. Immunization with the natural products induced high-titer antibodies against natural 9-O-acetyl-GD3 as well as F2 and F3. In contrast, mice immunized with the synthetic fractions produced antibodies only against the immunogen but not against natural 9-O-acetyl-GD3. Only immunization with the natural products induced production of antibodies reactive with surface antigens of melanoma cells expressing 9-O-acetyl-GD3. The findings suggest (a) that C-9 of the subterminal sialic acid is the site of chemical O-acetylation in F2 and F3, as opposed to C-9 of the terminal sialic acid in the natural products; (b) that O-acetylation of both the terminal and subterminal sialic acid moieties of GD3 results in recognition by three murine monoclonal antibodies (D1.1, ME 311, and Jones) reactive with human melanoma cells; (c) that O-acetylation of the terminal sialic acid is critical, on the other hand, for inducing an immune response against melanoma 9-O-acetyl-GD3; and (d) that O-acetyl GD3 from bovine buttermilk can substitute as immunogen for inducing an immune response against human melanoma cell surface antigens in the mouse.

Acetylation↗
PubMed1975

3-Hydroxy-3-methylglutaryl coenzyme A synthase. Evidence for an acetyl-S-enzyme intermediate and identification of a cysteinyl sulfhydryl as the site of acetylation.

Homogeneous liver 3-hydroxy-3-methylglutaryl coenzyme A synthase, which catalyzes the condensation of acetyl-CoA with acetoacetyl-CoA to form 3-hydroxy-3-methylglutaryl-CoA, also carries out: (a) a rapid transacetylation from acetyl-CoA to 31-dephospho-CoA and (b) a slow hydrolysis of acetyl-CoA to acetate and CoA. Transacetylation and hydrolysis occur at 50 and 1 percent, respectively, the rate of the synthasecatalyzed condensation reaction. It appears that an acetyl-enzyme intermediate is involved in the transacetylase and hydrolase reactions of 3-hydroxy-3-methylglutaryl-CoA synthase, as well as in the over-all condensation process. Covalent binding to the enzyme of a [14C]acetyl group contributed by [1(-14)C]acetyl-CoA is indicated by migration of the [14C]acetyl group with the dissociated synthase upon electrophoresis in dodecyl sulfate-urea and by precipitation of [14C]acetyl-enzyme with trichloroacetic acid. At 0 degrees and a saturating level of acetyl-CoA, the synthase is rapidly (less than 20 s) acetylated yielding 0.6 acetyl group/enzyme dimer. Performic acid oxidation completely deacetylates the enzyme, suggesting the site of acetylation to be a cysteinyl sulfhydryl group. Proteolytic digestion of [14C]acetyl-S-enzyme under conditions favorable for intramolecular S to N acetyl group transfer quantitatively liberates a labeled derivative with a [14C]acetyl group stable to performic acid oxidation. The labeled oxidation product is identified as N-[14C]acetylcysteic acid, thus demonstrating a cysteinyl sulfhydryl group as the original site of acetylation. The ability of the acetylated enzyme, upon addition of acetoacetyl-CoA, to form 3-hydroxy-3-methylglutaryl-CoA indicates that the acetylated cysteine residue is at the catalytic site.

Acetylation↗
PubMed1986

Nonrandom utilization of acetylation sites in histones isolated from Tetrahymena. Evidence for functionally distinct H4 acetylation sites.

Macro- and micronuclei of the ciliated protozoan Tetrahymena thermophila afford a unique opportunity to study histone acetylation under conditions where postsynthetic "transcription"-related acetylation and synthetic "deposition"-related acetylation are nonoverlapping. Recent studies have demonstrated that at least two general systems of acetylation operate in Tetrahymena. One is postsynthetic, macronuclear specific, and may be related to gene expression in that nucleus (Vavra, K. J., Allis, C. D., and Gorovsky, M. A. (1982) J. Biol. Chem. 257, 2591-2598). The other is synthetic, common to macro- and micronuclei, and is likely related to histone deposition during replication (Allis, C. D., Chicoine, L. G., Richman, R., and Schulman, I. G. (1985a) Proc. Natl. Acad. Sci. U. S. A., 82, 8048-8052). A unique feature of H3 and H4 in Tetrahymena is that neither are blocked at their amino termini. We have exploited this fact as well as the resolving capability of acid-urea gel electrophoresis and current microsequencing techniques to examine whether utilization of different NH2-terminal acetylation sites is random or nonrandom during the progression toward high acetylation states. Of the four acetylation sites which have been identified in H4 (in Tetrahymena these are lysines at positions 4, 7, 11, and 15), we find that lysine 7 is the exclusive site of postsynthetic acetylation in populations of monoacetylated H4 isolated from macronuclei. This site is retained in populations of diacetylated H4, which are acetylated exclusively at lysines 4 and 7. Our data also suggest that there is some preference for using lysine 11 (as compared to 15) as the third site of acetylation in triacetylated molecules. The data demonstrate that the postsynthetic acetylation-deacetylation process is surprisingly nonrandom for H4 in Tetrahymena macronuclei. We have also investigated the same question with macronuclear H3 (which contains acetylation sites at lysines 9, 14, 18, and 23). Our data demonstrate that unlike H4, lysines at position 9 or 14 are likely to be utilized as sites of acetylation within a population of monoacetylated H3. Both of these acetylation sites are retained in diacetylated H3 which suggests that if site 9 is used initially as the site of monoacetylation, 14 is used secondarily (and vice versa). Our data show, moreover, that there is a preference for utilizing lysine 18 as the third acetylation site (in triacetylated H3). Thus, these data show that H3 is also acetylated in a nonrandom fashion in macronuclei. Finally, we have determined which acetylation sites are utilized in macro- or micronuclear H4 when it is undergoing active synthesis and deposition.(ABSTRACT TRUNCATED AT 400 WORDS)

Acetylation↗
PubMed2005

On the mechanism of sequence-specific DNA-dependent acetylation of p53: the acetylation motif is exposed upon DNA binding.

P53 acetylation requires p300-docking to two contiguous sites in the activation domain that in turn mediates DNA-dependent acetylation of the tetramer. In an attempt to further define the mechanism of DNA-dependent acetylation of p53, an in vitro system has been reconstituted with distinct p53 isoforms and has been used to reveal conformational constraints on p53 acetylation. Two native p53 tetrameric isoforms purified from Sf9 cells differing by the extent of phosphorylation within the C-terminal acetylation site are both acetylated in a sequence-specific DNA-dependent manner. By contrast, p53 purified from an Escherichia coli expression system is in a largely denatured conformation and its acetylation is DNA-independent. Heating native p53 to destroy the folded structure restores DNA-independent acetylation similar to that seen with bacterially expressed p53. There are at least two sites of conformational flexibility in the p53 tetramer: the first in the flexible S10 beta-sheet within the MDM2 ubiquitination sequence and the second in the C-terminal regulatory domain. We analysed therefore whether DNA-dependent acetylation correlated with conformational changes in either of these two regions. DNA-dependent acetylation of p53 is maintained in a dose-dependent manner by low concentrations of consensus site DNA under conditions where flexibility in the S10 beta-sheet region is maintained. Oligonucleotide DNAs that promote acetylation stimulate the binding of monoclonal antibodies PAb421 and ICA-9; two antibodies whose contiguous epitopes overlap the C-terminal acetylation motif. By contrast, bent oligonucleotide DNAs that conceal both the S10 beta-sheet from binding of the monoclonal antibody DO-12 and attenuate binding of the monoclonal antibody PAb421 can preclude acetylation. These data suggest that, in the absence of DNA, the acetylation motif of p53 is in a cryptic state, but after DNA binding, allosteric effects mediate an exposure of the acetylation motif to allow DNA-dependent acetylation of the tetramer.

Acetylation↗
PubMed1987

Acetylator genotype-dependent metabolic activation of carcinogenic N-hydroxyarylamines by S-acetyl coenzyme A-dependent enzymes of inbred hamster tissue cytosols: relationship to arylamine N-acetyltransferase.

A genetic polymorphism in S-acetyl coenzyme A (AcCoA)-dependent N-acetyltransferase has been associated with a differential risk for certain cancers in humans. In this study, several tissues from the inbred Syrian hamster with a genetically defined AcCoA-dependent N-acetyltransferase polymorphism (homozygous rapid acetylator, Bio. 87.20; homozygous slow acetylator, Bio. 82.73/H; and heterozygous acetylator, Bio. 87.20 X Bio. 82.73/H F1), were investigated for the relationship of arylamine N-acetyltransferase to the AcCoA-dependent metabolic activation of carcinogenic N-hydroxy (N-OH)-arylamines to bind to DNA (O-acetyltransferase). The levels of both 2-aminofluorene (AF) N-acetyltransferase and N-OH-AF O-acetyltransferase activity reflected the N-acetylator genotype in liver, intestine, kidney and lung cytosols. A significant acetylator gene--dose response for AF N-acetyltransferase and N-OH-AF O-acetyltransferase activities was observed in liver and lung cytosols. In contrast, acetylator genotype was not consistently expressed for the AcCoA-dependent N-acetylation of 4-aminobiphenyl (ABP), nor for the AcCoA-dependent metabolic activation of N-OH-ABP and N-OH-3,2'-dimethyl-4-aminobiphenyl in these same tissue cytosols. Two peaks of acetyltransferase activity were partially purified by ion exchange FPLC chromatography from the hepatic cytosol of both the homozygous rapid and homozygous slow acetylator hamster. In contrast to unfractionated cytosol, the isozyme(s) eluting first clearly demonstrated levels of AcCoA-dependent arylamine N-acetyltransferase and N-OH-arylamine O-acetyltransferase activities that were consistent with N-acetylator genotype (polymorphic) for all substrates tested. In contrast, the slower eluting isozyme(s) in each acetylator cytosol showed levels of AcCoA-dependent N- and O-acetyltransferase activities that did not vary with N-acetylator genotype (monomorphic). The AcCoA-dependent O-acetyltransferase activity of both the monomorphic and polymorphic peaks was paraoxon resistant. These studies demonstrate acetylator genotype-dependent control of AcCoA-dependent metabolic activation of N-OH-arylamines(O-acetylation) by polymorphic isozyme(s) similar to that for AcCoA-dependent N-acetylation of arylamines in the hamster. The polymorphic genetic control of N-OH-arylamine O-acetyltransferase may be an important risk factor for arylamine-induced cancer, in those species and tissues expressing appreciable levels of O-acetyltransferase activity.

Acetyl Coenzyme A↗
PubMed1985

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

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

Acetyl Coenzyme A↗