Reactive Thioester Research Articles

Studies on the reactivity of clofibryl-S-acyl-CoA thioester with glutathione in vitro.

Clofibric acid (p-chlorophenoxyisobutyric acid) is metabolized in vivo to a thioester-linked glutathione conjugate, S-(p-chlorophenoxyisobutyryl)glutathione (CA-SG). The formation of this metabolite is presumed to occur via transacylation reactions between glutathione (GSH) and reactive acyl-linked metabolite(s) of the drug. The present study examines the chemical reactivity of clofibryl-S-acyl-CoA (CA-SCoA), an acyl-CoA thioester intermediary metabolite of clofibric acid, with GSH to form the CA-SG in vitro. Incubations of CA-SCoA (1 mM) with GSH (5 mM) were carried out at pH 7.5 and 37 degrees C, with analysis of the formed reaction products by isocratic reverse-phase high-performance liquid chromatography (HPLC). Results showed a time-dependent and linear formation of CA-SG up to 4 h (50 microM CA-SG formed/h), and after a 1-day incubation, the reaction mixture contained 0.7 mM CA-SG. The identity of CA-SG was confirmed by analysis of HPLC-purified material by tandem mass spectrometry. The rate of CA-SG formation was found to be increased 3-fold in incubations containing rat liver glutathione S-transferases (4 mg/ml). Analysis of the chemical stability of CA-SCoA in buffer at 37 degrees C and varying pH showed the derivative to be stable under mildly acidic and basic aqueous conditions but to hydrolyze at pH values greater than 10 after a 1-day incubation (t(1/2) = approximately 1 day at pH 10.5). Results from these studies show that CA-SCoA is a reactive thioester derivative of clofibric acid and is able to acylate GSH and other thiol-containing nucleophiles in vitro and, therefore, may be able to acylate protein thiols in vivo, which could contribute to the toxic side effects of the drug.

Methanopyrus kandleri Glutamyl-tRNA Reductase

The initial reaction of tetrapyrrole formation in archaea is catalyzed by a NADPH-dependent glutamyl-tRNA reductase (GluTR). The hemA gene encoding GluTR was cloned from the extremely thermophilic archaeon Methanopyrus kandleri and overexpressed in Escherichia coli. Purified recombinant GluTR is a tetrameric enzyme with a native M(r) = 190,000 +/- 10,000. Using a newly established enzyme assay, a specific activity of 0.75 nmol h(-1) mg(-1) at 56 degrees C with E. coli glutamyl-tRNA as substrate was measured. A temperature optimum of 90 degrees C and a pH optimum of 8.1 were determined. Neither heme cofactor, nor flavin, nor metal ions were required for GluTR catalysis. Heavy metal compounds, Zn(2+), and heme inhibited the enzyme. GluTR inhibition by the newly synthesized inhibitor glutamycin, whose structure is similar to the 3' end of the glutamyl-tRNA substrate, revealed the importance of an intact chemical bond between glutamate and tRNA(Glu) for substrate recognition. The absolute requirement for NADPH in the reaction of GluTR was demonstrated using four NADPH analogues. Chemical modification and site-directed mutagenesis studies indicated that a single cysteinyl residue and a single histidinyl residue were important for catalysis. It was concluded that during GluTR catalysis the highly reactive sulfhydryl group of Cys-48 acts as a nucleophile attacking the alpha-carbonyl group of tRNA-bound glutamate with the formation of an enzyme-localized thioester intermediate and the concomitant release of tRNA(Glu). In the presence of NADPH, direct hydride transfer to enzyme-bound glutamate, possibly facilitated by His-84, leads to glutamate-1-semialdehyde formation. In the absence of NADPH, a newly discovered esterase activity of GluTR hydrolyzes the highly reactive thioester of tRNA(Glu) to release glutamate.

Crystal structure of the histone acetyltransferase domain of the human PCAF transcriptional regulator bound to coenzyme A.

The human p300/CBP-associating factor, PCAF, mediates transcriptional activation through its ability to acetylate nucleosomal histone substrates as well as transcriptional activators such as p53. We have determined the 2.3 A crystal structure of the histone acetyltransferase (HAT) domain of PCAF bound to coenzyme A. The structure reveals a central protein core associated with coenzyme A binding and a pronounced cleft that sits over the protein core and is flanked on opposite sides by the N- and C-terminal protein segments. A correlation of the structure with the extensive mutagenesis data for PCAF and the homologous yeast GCN5 protein implicates the cleft and the N- and C-terminal protein segments as playing an important role in histone substrate binding, and a glutamate residue in the protein core as playing an essential catalytic role. A structural comparison with the coenzyme-bound forms of the related N-acetyltransferases, HAT1 (yeast histone acetyltransferase 1) and SmAAT (Serratia marcescens aminoglycoside 3-N-acetyltransferase), suggests the mode of substrate binding and catalysis by these enzymes and establishes a paradigm for understanding the structure-function relationships of other enzymes that acetylate histones and transcriptional regulators to promote activated transcription.

Semisynthesis of cytotoxic proteins using a modified protein splicing element.

Two cytotoxic proteins, bovine pancreatic ribonuclease A (RNase A), and a restriction endonuclease from Haemophilus parainfluenzae (HpaI), were produced using a novel semisynthetic approach that utilizes a protein splicing element, an intein, to generate a reactive thioester at the C-terminus of a recombinant protein. Nucleophilic attack on this thioester by the N-terminal cysteine of a synthetic peptide ultimately leads to the ligation of the two reactants through a native peptide bond. This strategy was used to produce RNase A and HpaI by isolating inactive truncated forms of these proteins, the first 109 and 223 amino acids of RNase A and HpaI, respectively, as fusion proteins consisting of the target protein, an intein, and a chitin binding domain. Thiol-induced cleavage of the precursor led to the liberation of the target protein with a C-terminal thioester-tag. Addition of synthetic peptides representing the amino acids missing from the truncated forms led to the generation of full-length products that displayed catalytic activity indicative of the wild-type enzymes. The turnover numbers and Km for ligated and renatured RNase A were 8.2 s(-1) and 1.5 mM, in good agreement with reported values of 8.3 s(-1) and 1.2 mM (Hodges & Merrifield, 1975). Ligated HpaI had a specific activity of 0.5-1.5 x 10(6) U/mg, which compared favorably with the expected value of 1-2 x 10(6) U/mg (J. Benner, unpubl. obs.). Besides assisting in the production of cytotoxic proteins, this technique could allow the easy insertion of unnatural amino acids into a protein sequence.

Innate immunity.

Innate immunity.

Prebiotic amino acid thioester synthesis: thiol-dependent amino acid synthesis from formose substrates (formaldehyde and glycolaldehyde) and ammonia.

Formaldehyde and glycolaldehyde (substrates of the formose autocatalytic cycle) were shown to react with ammonia yielding alanine and homoserine under mild aqueous conditions in the presence of thiol catalysts. Since similar reactions carried out without ammonia yielded alpha-hydroxy acid thioesters (Weber, 1984a, b), the thiol-dependent synthesis of alanine and homoserine is presumed to occur via amino acid thioesters--intermediates capable of forming peptides (Weber and Orgel 1979). A pH 5.2 solution of 20 mM formaldehyde, 20 mM glycolaldehyde, 20 mM ammonium chloride, 23 mM 3-mercaptopropionic acid, and 23 mM acetic acid that reacted for 35 days at 40 degrees C yielded (based on initial formaldehyde) 1.8% alanine and 0.08% homoserine. In the absence of thiol catalyst, the synthesis of alanine and homoserine was negligible. Alanine synthesis required both formaldehyde and glycolaldehyde, but homoserine synthesis required only glycolaldehyde. At 25 days the efficiency of alanine synthesis calculated from the ratio of alanine synthesized to formaldehyde reacted was 2.1%, and the yield (based on initial formaldehyde) of triose and tetrose intermediates involved in alanine and homoserine synthesis was 0.3 and 2.1%, respectively. Alanine synthesis was also seen in similar reactions containing only 10 mM each of aldehyde substrates, ammonia, and thiol. The prebiotic significance of these reactions that use the formose reaction to generate sugar intermediates that are converted to reactive amino acid thioesters is discussed.

Modulation of Reactivity in Native Chemical Ligation through the Use of Thiol Additives

In native chemical ligation, an unprotected peptide α-carboxy thioester is reacted with a second peptide containing an N-terminal cysteine residue. It was anticipated that addition of thiophenol to a native chemical ligation reaction would keep cysteine side chains reduced, catalyze the reversal of unproductive thioester formation, and generate a more reactive phenyl thioester through thiol exchange. Several model peptide−α-thioesters were treated with an excess of a competing thiol to investigate their susceptibility to thiol exchange: a highly activated 3-nitro-4-carboxybenzyl α-thioester was smoothly converted to the less activated benzyl α-thioester through the addition of an excess of benzyl mercaptan; similarly, a peptide containing the benzyl α-thioester group was converted to a more reactive phenyl α-thioester by addition of thiophenol. Even a weakly activated peptide−α-thioester was converted to a substantially more reactive species, as demonstrated by the conversion of peptide−αCOS-CH2COOH to peptide−αCOS-phenyl. The utility of in situ transthioesterification in native chemical ligation reactions was demonstrated by model syntheses of the 110-residue barnase polypeptide chain. The use of thiophenol as an additive in the ligation gave clean, rapid reaction to form the desired amide-linked product in high yield. The in situ transthioesterification process is broadly applicable to the total chemical synthesis of proteins by native chemical ligation.

Myelin P0 Glycoprotein and a Synthetic Peptide Containing the Palmitoylation Site Are Both Autoacylated

P0, the major protein of the PNS myelin, is palmitoylated at the cytoplasmic Cys153. To gain insights into the mechanism of P0 acylation, the in vitro palmitoylation of both P0 and a synthetic Cys153-containing octapeptide was studied. Incubation of PNS myelin membranes or isolated P0 with [3H]palmitoyl-CoA resulted in specific labeling of this protein, suggesting that the reaction is nonenzymatic. Incorporation of the labeled fatty acid into P0 was not affected by boiling the isolated P0 for 15 min before incubation or by adding sciatic nerve homogenate to the reaction mixture, which confirms the nonenzymatic nature of the reaction. After chemical deacylation, P0 was palmitoylated at a higher rate, suggesting that the original site was reacylated. Furthermore, tryptic digestion and peptide mapping showed that the same sites are acylated in vitro as in nerve slices indicating that the reaction has physiological significance. On incubation with [14C]palmitoyl-CoA, the synthetic peptide encompassing the natural P0 acylation site (I150RYCWLRR157) was also spontaneously acylated at the cysteine residue. Thus, the integrity of the protein is not required for the nonenzymatic transacylation reaction. At pH 7.4 and 37 degrees C, peptide palmitoylation followed a second-order reaction (k2 = 246 +/- 6 M-1 min-1) and is likely a bimolecular nucleophilic substitution with the peptide thiolate attacking the highly reactive thioester bond in palmitoyl-CoA. The activation energy calculated from the Arrhenius plot is approximately 2 kcal/mol and much lower than that of enzyme-catalyzed transacylations. Finally, two other P0 peptides (V121PTRYG126 and K109TSQVTL115) as well as various unrelated thiol-containing compounds, including cysteine, glutathione, pressinoic acid (CYFQNC), and crustacean cardioactive peptide (PFCNAFTGC), were not autoacylated. These results indicate that the IRYCWLRR peptide represents a particular structural motif and/or has some chemical features that allow the reaction to occur spontaneously.

Covalent attachment of human complement C3 to IgG. Identification of the amino acid residue involved in ester linkage formation.

C3 (native complement component 3) plays a central role in the activation of complement and in the transport and processing of immune complexes. Proteolytic activation of C3 exposes a highly reactive thioester bond which preferentially reacts with the hydroxyl groups of acceptor molecules on activators such as immune complexes or carbohydrates on microorganisms. Recently, a C3 attachment site has been localized on the CH1 domain of IgG1 between Val134 and Lys156. We have synthesized a series of peptide analogs of this region to identify the preferred residue for C3b (the proteolytically activated form of C3) attachment. The parent peptide included all 6 hydroxyl-containing amino acids present in the proposed binding site and was highly reactive with activated C3. The C3b-peptide complex was sensitive to hydroxylamine as was C3b-IgG indicating that both were ester-linked. The kinetic profile of hydrolysis of the C3b-peptide complex under physiologic conditions was found to be nearly identical to the profiles of C3b-IgG, C3b-IgG1, and C3b-glycerol complexes. Site-specific amino acid substitution of threonine and serine residues in the peptide indicated that, in contrast to the attachment site in C4b, little or no attachment occurred at serine residues. The threonine corresponding to Thr144 in the CH1 domain of IgG was found to be the major acceptor site for C3b. Thr148 was the second most reactive site on the peptide, but this residue is buried in native IgG.

Purification of chicken C3 and a structural and functional characterization.

A major plasma protein from chicken, analogous to mammalian complement component C3, was purified by the removal of plasminogen, precipitation with polyethyleneglycol, and ion-exchange chromatography. Purification was guided by a rabbit antiserum specific to chicken C3. The yield of native C3 was 27%, and purity and functional activity was assessed by SDS-PAGE, immunoprecipitation techniques, and the ability of the purified C3 to restore the haemolytic activity of C3-depleted chicken serum. Monoclonal antibodies were raised against purified chicken C3. These antibodies were characterized and used to prepare an immunosorbent column to deplete chicken plasma specifically of C3. Chicken C3 has a mol.wt of 185,000-195,000 and a two-chain structure with an alpha chain (118,000) and beta chain (68,000). Complement activation leads to changes in the electrophoretic mobility of chicken C3 and to a decrease in mol.wt to 144,000 corresponding to the release of a 15,000 C3a and a 34,000 C3d/C3dg fragment. Chicken C3 exists in multiple molecular forms with pI values of 6.4-6.6. A genetic polymorphism of chicken C3 based on electrophoretic mobility has not yet been detected after analysis of more than 500 individuals. The function of chicken C3 is dependent on a reactive thioester because treatment of purified chicken C3 with methylamine causes functional inactivation of C3.

Spin-label and fluorescence labeling studies of the thioester bonds in human .alpha.2-macroglobulin

Upon cleavage of the reactive thioester bonds (Cys-949-Glx-952) of tetrameric human alpha 2-macroglobulin (alpha 2M) by methylamine, one sulfhydryl group per alpha 2M subunit is exposed. These identical sulfhydryl group sites were labeled with the thiol-specific nitroxide spin-labels (1-oxy-2,2,5,5-tetramethyl-3-pyrrolin-3-yl)methyl methanethiosulfonate and (1-oxy-2,2,6,6-tetramethyl-4-piperidinyl)methyl methanethiosulfonate, a homologous series of maleimide spin-labels, and the thiol-specific fluorescent probe 2-[(4-maleimidophenyl)amino]naphthalene-6-sulfonic acid sodium salt (MANS). The ESR and fluorescence results showed that these sulfhydryl group sites were at the base of a narrow crevice that is greater than or equal to 8 A deep. Although the bound MANS fluorophore was slightly blue shifted with an enhanced quantum yield vs the free label in water, the environment of the sulfhydryl site appeared to be of a polar nature when compared with the emission maxima in several solvents of varying polarity. The Glx residue participating in the thioester linkage in the intact protein was labeled with 4-amino-2,2,6,6-tetramethylpiperidine-1-oxyl. The distance between the Glx and Cys moieties was estimated at greater than or equal to 10-25 A from double spin-labeling experiments.

Clostridium histolyticum collagenase: development of new thio ester, fluorogenic, and depsipeptide substrates and new inhibitors.

A new series of thio ester, depsipeptide, and peptide substrates have been synthesized for the bacterial enzyme Clostridium histolyticum collagenase. The hydrolysis of the depsipeptide substrate was followed on a pH stat, and thio ester hydrolysis was measured by inclusion of the chromogenic thiol reagent 4,4'-dithiopyridine in the assay mixture. The best thio ester substrate, Boc-Abz-Gly-Pro-Leu-SCH2CO-Pro-Nba, had a kcat/KM of 63 000 M-1 s-1, while several shorter thio ester sequences were inactive as substrates. In general, the peptide analogues of all the reactive thio ester substrates were shown to be hydrolyzed 5-10 times faster by collagenase. In one case (Z-Gly-Pro-Leu-Gly-Pro-NH2) where a comparison was made, the peptide substrate was respectively 8- and 106-fold more readily hydrolyzed than the corresponding thio ester and ester substrates. Cleavages of the two fluorescence-quench substrates Abz-Gly-Pro-Leu-Gly-Pro-Nba and Abz-Gly-Pro-Leu-SCH2CO-Pro-Nba could be easily followed fluorogenically since a 5-10-fold increase in fluorescence occurred upon hydrolysis. The fluorescent peptide substrate is the best synthetic substrate known for C. histolyticum collagenase with a kcat/KM value of 490 000 M-1 s-1. A series of new reversible inhibitors were developed by the attachment of zinc ligating groups (hydroxamic acid, carboxymethyl, and thiol) to various peptide sequences specific for C. histolyticum collagenase. The shorter peptides designed to bind to either the P3-P1 or P1'-P3' subsites were poor to moderate inhibitors. The thiol HSCH2CH2CO-Pro-Nba had the lowest K1 (0.02 mM).(ABSTRACT TRUNCATED AT 250 WORDS)

Gramicidin S synthetase. Stability of reactive thioester intermediates and formation of 3-amino-2-piperidone.

The reactive thioester complexes of gramicidin S synthetase with substrate amino acids and intermediate peptides are slowly hydrolyzed in neutral buffer solutions under mild conditions. Fully active enzyme is recovered. These processes are strongly accelerated by certain thiol protective agents. In the presence of 1 mM dithioerythritol the half-life times of these hydrolysis reactions are in the range of 1-90 h at 3 degrees C. The thioester complex of gramicidin S synthetase 2 (GS2, the heavy enzyme) with the tripeptide DPhe-Pro-Val is distinguished by the highest stability of all these intermediates. A different decomposition pattern is observed for the thioester complex of GS2 with LOrn. Here 3-amino-2-piperidone (cyclo-LOrn) is formed in a rapid cyclization reaction. This product specifically blocks the activation center of GS2 for LOrn at the thioester binding site. All other activation reactions of gramicidin S synthetase are unaffected. A procedure for a specific labelling of the reaction centers of the multienzyme is outlined.