Acetyl-enzyme Intermediate Research Articles

The Polysialic Acid-specific O-Acetyltransferase OatC from Neisseria meningitidis Serogroup C Evolved Apart from Other Bacterial Sialate O-Acetyltransferases

Neisseria meningitidis serogroup C is a major cause of bacterial meningitis and septicaemia. This human pathogen is protected by a capsule composed of alpha2,9-linked polysialic acid that represents an important virulence factor. In the majority of strains, the capsular polysaccharide is modified by O-acetylation at C-7 or C-8 of the sialic acid residues. The gene encoding the capsule modifying O-acetyltransferase is part of the capsule gene complex and shares no sequence similarities with other proteins. Here, we describe the purification and biochemical characterization of recombinant OatC. The enzyme was found as a homodimer, with the first 34 amino acids forming an efficient oligomerization domain that worked even in a different protein context. Using acetyl-CoA as donor substrate, OatC transferred acetyl groups exclusively onto polysialic acid joined by alpha2,9-linkages and did not act on free or CMP-activated sialic acid. Motif scanning revealed a nucleophile elbow motif (GXS286XGG), which is a hallmark of alpha/beta-hydrolase fold enzymes. In a comprehensive site-directed mutagenesis study, we identified a catalytic triad composed of Ser-286, Asp-376, and His-399. Consistent with a double-displacement mechanism common to alpha/beta-hydrolase fold enzymes, a covalent acetylenzyme intermediate was found. Together with secondary structure prediction highlighting an alpha/beta-hydrolase fold topology, our data provide strong evidence that OatC belongs to the alpha/beta-hydrolase fold family. This clearly distinguishes OatC from all other bacterial sialate O-acetyltransferases known so far because these are members of the hexapeptide repeat family, a class of acyltransferases that adopt a left-handed beta-helix fold and assemble into catalytic trimers.

Identification of catalytically important amino acid residues of Streptomyces lividans acetylxylan esterase A from carbohydrate esterase family 4

Multiple sequence alignment of Streptomyces lividans acetylxylan esterase A and other carbohydrate esterase family 4 enzymes revealed the following conserved amino acid residues: Asp-12, Asp-13, His-62, His-66, Asp-130, and His-155. These amino acids were mutated in order to investigate a functional role of these residues in catalysis. Replacement of the conserved histidine residues by alanine caused significant reduction of enzymatic activity. Maintenance of ionizable carboxylic group in side chains of amino acids at positions 12, 13, and 130 seems to be necessary for catalytic efficiency. The absence of conserved serine excludes a possibility that the enzyme is a serine esterase, in contrast to acetylxylan esterases of carbohydrate esterase families 1, 5, and 7. On the contrary, total conservation of Asp-12, Asp-13, Asp-130, and His-155 along with dramatic decrease in enzyme activity of mutants of either of these residues lead us to a suggestion that acetylxylan esterase A from Streptomyces lividans and, by inference, other members of carbohydrate esterase family 4 are aspartic deacetylases. We propose that one component of the aspartate dyad/triad functions as a catalytic nucleophile and the other one(s) as a catalytic acid/base. The ester/amide bond cleavage would proceed via a double displacement mechanism through covalently linked acetyl-enzyme intermediate of mixed anhydride type.

Kinetic Studies on the Optical Resolution of 2-Alkanol by Pseudomonas cepaciaLipase Catalyzed Transesterification with Vinyl Acetate in Organic Solvent.

The optical resolution data were analyzed for Pseudomonas cepacia lipase (PCL) catalyzed transesterification between vinyl acetate and 2-alkanol in organic solvent. The plots of log E (E: the enantiomeric ratio as the reaction is irreversible) agaist the extent of conversion (c) showed the maxima for all systems used. The competitive reaction in the optical resolution was verified by the analysis of the resolution data and an accordance of the enantiomeric excess (ee) vs. c plots between racemic and optically pure alcohols. The reverse reaction in the optical resolution was experimentally proved by a decrease in optical purity for the reaction of optically pure 1-methylheptyl acetate and 2-octanol in the presence of PCL. The first order plot showed a lenearity for the reaction with (R)-2-octanol but curveture for the (S) counterpart. Based on the above observations, we concluded that the transesterification with vinyl acetate did not obey the reported optical resolution equations because acetylation of the (S) isomer in racemic 2-alkanol with the acetyl-enzyme intermediate was surpressed. Thus, E increased with c in the range of below 0.5.

Enzyme-Catalyzed Acylation of Homoserine: Mechanistic Characterization of the Haemophilus influenzae met2-Encoded Homoserine Transacetylase

The first unique step in bacterial and plant methionine biosynthesis involves the acylation of the gamma-hydroxyl of homoserine. In Haemophilus influenzae, acylation is accomplished via an acetyl-CoA-dependent acetylation catalyzed by homoserine transacetylase. The activity of this enzyme regulates flux of homoserine into multiple biosynthetic pathways and, therefore, represents a critical control point for cell growth and viability. We have cloned homoserine transacetylase from H. influenzae and present the first detailed enzymatic study of this enzyme. Steady-state kinetic experiments demonstrate that the enzyme utilizes a ping-pong kinetic mechanism in which the acetyl group of acetyl-CoA is initially transferred to an enzyme nucleophile before subsequent transfer to homoserine to form the final product, O-acetylhomoserine. The maximal velocity and V/K(homoserine) were independent of pH over the range of values tested, while V/K(acetyl)(-)(CoA) was dependent upon the ionization state of a single group exhibiting a pK value of 8.6, which was required to be protonated. Solvent kinetic isotope effect studies yielded inverse effects of 0.75 on V and 0.74 on V/K(CoA) on the reverse reaction and effects of 1.2 on V and 1.7 on V/K(homoserine) on the forward reaction. Direct evidence for the formation of an acetyl-enzyme intermediate was obtained using rapid-quench labeling studies. On the basis of these observations, we propose a chemical mechanism for this important member of the acyltransferase family and contrast its mechanism with that of homoserine transsuccinylase.

Redox-dependent acetyl transfer partial reaction of the acetyl-CoA decarbonylase/synthase complex: kinetics and mechanism.

Acetyl-CoA decarbonylase/synthase (ACDS) is a multienzyme complex that plays a central role in energy metabolism in Methanosarcina barkeri grown on acetate. The ACDS complex carries out an unusual reaction involving net cleavage of the acetyl C-C and thioester bonds of acetyl-CoA. The overall reaction is composed of several partial reactions, one of which involves catalysis of acetyl group transfer. To gain insight into the overall reaction, a study was carried out on the kinetics and mechanism of the acetyltransferase partial reaction. Analysis by HPLC was used to quantify rates of acetyl transfer from acetyl-CoA both to 3'-dephospho-CoA and, by isotope exchange, to 14C-labeled CoA. Acetyl transfer activity was observed only under strongly reducing conditions, and was half-maximal at -486 mV at pH 6.5. The midpoint activation potential became increasingly more negative as the pH was increased, indicating the involvement of a protonation step. Cooperative dependence on acetyl-CoA concentration was exhibited in reactions that contained incompletely reduced enzyme; however, under redox conditions supporting maximum activity, hyperbolic kinetics were found. A ping-pong steady state kinetic mechanism was established, consistent with formation of an acetyl-enzyme intermediate. Analysis of the inhibitory effects of CoA on acetyl transfer to 3'-dephospho-CoA provided values for KiCoA of 6.8 microM and for Kiacetyl-CoA of 45 microM; isotope exchange analyses yielded values of 32 and 120 microM, respectively. Two separate measures of stability yielded values for the free energy of hydrolysis of the acetyl-enzyme intermediate of -9.6 and -9.3 kcal/mol, an indication of a high-energy bonding interaction in the acetyl-enzyme species. Implications for the mechanism of C-C bond cleavage are discussed.

Mechanism of action of clostridial glycine reductase: isolation and characterization of a covalent acetyl enzyme intermediate

Clostridial glycine reductase consists of proteins A, B, and C and catalyzes the reaction glycine + Pi + 2e(-)----acetyl phosphate + NH4+. Evidence was previously obtained that is consistent with the involvement of an acyl enzyme intermediate in this reaction. We now demonstrate that protein C catalyzes exchange of [32P]Pi into acetyl phosphate, providing additional support for an acetyl enzyme intermediate on protein C. Furthermore, we have isolated acetyl protein C and shown that it is qualitatively catalytically competent. Acetyl protein C can be obtained through the forward reaction from protein C and Se-(carboxymethyl)selenocysteine-protein A, which is generated by the reaction of glycine with proteins A and B [Arkowitz, R. A., & Abeles, R. H. (1990) J. Am. Chem. Soc. 112, 870-872]. Acetyl protein C can also be generated through the reverse reaction by the addition of acetyl phosphate to protein C. Both procedures lead to the same acetyl enzyme. The acetyl enzyme reacts with Pi to give acetyl phosphate. When [14C]acetyl protein C is denaturated with TCA and redissolved with urea, radioactivity remained associated with the protein. At pH 11.5 radioactivity was released with t1/2 = 57 min, comparable to the hydrolysis rate of thioesters. Exposure of 4 N neutralized NH2OH resulted in the complete release of radioactivity. Treatment with KBH4 removes all the radioactivity associated with protein C, resulting in the formation of [14C]ethanol. We conclude that a thiol group on protein C is acetylated. Proteins A and C together catalyze the exchange of tritium atoms from [3H]H2O into acetyl phosphate.(ABSTRACT TRUNCATED AT 250 WORDS)

Purification and characterization of [acyl-carrier-protein] acetyltransferase from Escherichia coli.

A multi-step procedure has been developed for the purification of [acyl-carrier-protein] acetyltransferase from Escherichia coli, which allows the production of small amounts of homogeneous enzyme. The subunit Mr was estimated to be 29,000 and the native Mr was estimated to be 61,000, suggesting a homodimeric structure. The catalytic properties of the enzyme are consistent with a Bi Bi Ping Pong mechanism and the existence of an acetyl-enzyme intermediate in the catalytic cycle. The enzyme was inhibited by N-ethylmaleimide and more slowly by iodoacetamide in reactions protected by the substrate, acetyl-CoA. However, the enzyme was apparently only weakly inhibited by the thiol-specific reagent methyl methanethiosulphonate. The nature of the acetyl-enzyme intermediate is discussed in relationship to that found in other similar enzymes from E. coli, yeast and vertebrates.

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.

Substituent effects on substrate activation and Michaelis-Menten Kinetic parameters in the alpha-chymotrypsin-catalyzed hydrolysis of phenyl acetates.

The effects of substituents on the steady state and pre-steady state kinetics in alpha-chymotrypsin [EC 3.4.21.1]-catalyzed hydrolysis were studied using substituted phenyl acetates. In the steady state hydrolysis, substrate activation, which had been observed and studied previously for p-nitrophenyl acetate, was also observed for p-bromo, p-chloro-, and m-methylphenyl acetates. Little activation was observed for p-acetyl-, m-nitro-, p-methyl-, and p-methoxyphenyl acetates. Addition of p-dichlorobenzene increased kcat for all substrates examined and greatly diminished the substrate activation for the activatable substrate(s) to activator binding site(s). The value of kcat decreased in accordance with increase of the sigma-value of substituents. On the other hand, kcat/Km (app) showed an opposite sigma- dependence, as was previously observed. In pre-steady state measurements, little burst was observed for more electron-donating substituents than m-nitro. The sigma dependence of kcat is apparently not consistent with the prediction derived from that of kcat/Km (app) on the basis of the usual two-step mechanism with a common acetyl-enzyme intermediate.

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.

Acetyl Coenzyme A:Arylamine Acetyltransferase

Abstract A preparation of acetyl-CoA:arylamine N-acetyltransferase from pigeon liver is rapidly and completely inactivated by iodoacetamide with a second order rate constant of 1950 m-1 min-1 at 25°, pH 7.5. This inactivation is prevented by p-nitrophenyl acetate, an active substrate, at concentrations as low as 1.7 x 10-8 m, far below the Ks value of 8.6 x 10-3 m. The protection afforded by 1.67 x 10-3 m p-nitrophenyl acetate is removed to varying extents in the presence of acceptor anilines under conditions in which the enzyme is catalyzing acetyl transfer. Saturating concentrations of p-toluidine bring about complete removal of protection, whereas saturating concentrations of p-cyanoaniline bring about only a 19% removal of protection. This effect of anilines exhibits the same dependence upon aniline concentration as does the rate of the normal catalyzed reaction. It is concluded: (a) there is a kinetically significant acetyl-enzyme intermediate that is not subject to inactivation by iodoacetamide; (b) the acetyl-enzyme intermediate is almost certainly a thiol ester; (c) the rate of inactivation by iodoacetamide provides a probe for the fraction of enzyme that exists as acetyl-enzyme during the steady state turnover of substrates; (d) with p-nitrophenyl acetate and p-toluidine acetyl-enzyme formation is the rate-determining step, whereas with the less reactive acceptor p-cyanoaniline the deacylation step is rate-determining, confirming earlier conclusions from kinetic data.

Acetylcholinesterase: New evidence for an acetyl-enzyme intermediate

Acetylcholinesterase: New evidence for an acetyl-enzyme intermediate