Nucleophilic Acceptor Research Articles

Cobalt(III)-ligated peptides as acyl acceptors in peptide synthesis catalyzed by chymotrypsin

The efficiency of cobalt(III)-ligated peptides as acceptor nucleophiles in acyl transfer reactions catalyzed by α-chymotrypsin was examined. A series of metallopeptides with the general formula [H-(Gly) n -OCo(NH 3) 5] 2+ (1 ≤ n ≤ 4) was tested. The aminolysis rate of acyl-chymotrypsin was measured spectrophotometrically by monitoring the concentration of unreacted nucleophile. The rate of the competing hydrolysis of acyl-chymotrypsin was obtained by automatic titration with base, using a pH-stat. The main result was that the positively charged metallopeptides, in general, were more efficient nucleophiles than the corresponding amides and free peptides that were examined for comparison. A binding model that rationalizes the findings is given.

Biotinylation of biotinidase following incubation with biocytin

Human serum biotinidase, purified to homogeneity (1920 units/mg protein), was incubated with biocytin prior to electrophoresis and transblotting with avidin-peroxidase. Avidin reacted with biotinidase maximally when incubated at pH 7.5–9, less at pH 7 and none below pH 7. No avidin reactivity occurred when biotinidase was incubated with biotin or in the absence of biocytin. Inclusion of the nucleophilic acceptors, ethanolamine or hydroxylamine, to the incubation mixture with biocytin and biotinidase resulted in loss of avidin reactivity. High concentrations of mercaptoethanol also prevented avidin reactivity. These results suggest that biotinidase can be biotinylated in the presence of biocytin at neutral to alkaline pH probably through a thioester bond formed with a cysteine residue in the active site of the enzyme. Biotinidase may then function as a biotinylating enzyme when incubated with appropriate nucleophilic acceptors.

Coupling in the absence of tertiary amines. V. Removal of the 2-nitrobenzenesulfenyl (NPS) group with 1-hydroxybenzotriazole in the presence of aniline or N-methylaniline.

In a search for conditions of acidolytic removal of amine protecting groups leading to salts of the deblocked amine that can be acylated without addition of a tertiary amine, cleavage of the 2-nitrobenzenesulfenyl (Nps) group with hydroxybenzotriazole (HOBt) in 2,2,2-trifluoroethanol was attempted. The Nps group was smoothly removed, but the resulting salt of the amine component could not be acylated unless deprotonated with a tertiary base. A rationale is now proposed for this unsatisfactory outcome of the cleavage reaction and for the concomitant surprising reduction of HOBt to benzotriazole. Based on the proposed mechanism, a new approach was designed for the removal of the Nps group. It was cleaved with HOBt in the presence of weakly basic nucleophiles such as aniline, N-methylaniline or 8-aminoquinoline. The protecting group was transferred smoothly to the amino group of the nucleophilic acceptor leaving the deblocked amine component in the form of its HOBt salt. This was then readily acylated without addition of a tertiary amine.

A new kinetic mechanism for the concomitant hydrolysis and transfer reactions catalyzed by bacterial DD-peptidases.

In the presence of an adequate nucleophilic acceptor substrate (A) and ester and thiolester donor substrates (S), the Streptomyces R61 soluble DD-peptidase catalyzes both hydrolysis and acyl group transfer reactions. Simple bisubstrate models do not explain the variation of the transfer to hydrolysis ratios with the donor and acceptor concentrations. A new kinetic mechanism for the concomitant hydrolysis and transfer reactions is proposed which involves an acceptor and a second, nonproductive donor substrate binding site. In this model, the acceptor essentially binds to the acyl-enzyme, and the second donor molecule only binds to the ternary ES*A complex. Hydrolysis can then proceed from the quaternary ES*AS complex. The values of all of the parameters involved in the reaction of a thiolester substrate with D-alanine as the acceptor substrate were determined at 15 and 37 degrees C. The results obtained with a protein modified by site-directed mutagenesis, and with which the transpeptidation reaction appeared to be specifically impeded, are discussed on the basis of the new kinetic mechanism. The data obtained with the soluble form of the high molecular weight penicillin binding protein 2X from Streptococcus pneumoniae are also in agreement with this model.

Overexpression, site-directed mutagenesis, and mechanism of Escherichia coli acid phosphatase

Site-directed mutagenesis was used to examine the catalytic importance of 2 histidine and 4 arginine residues in Escherichia coli periplasmic acid phosphatase (EcAP). The residues that were selected as targets for mutagenesis were those that were also conserved in a number of high molecular weight acid phosphatases from eukaryotic organisms, including human prostatic and lysosomal acid phosphatases. Both wild type EcAP and mutant proteins were overproduced in E. coli using an expression system based on the T7 RNA polymerase promoter, and the proteins were purified to homogeneity. Examination of the purified mutant proteins by circular dichroism and proton NMR spectroscopy revealed no significant conformational changes. The replacement of Arg16 and His17 residues that were localized in a conserved N-terminal RHGXRXP motif resulted in the complete elimination of EcAP enzymatic activity. Critical roles for Arg20, Arg92, and His303 were also established because the corresponding mutant proteins exhibited residual activities that were not higher than 0.4% of that of wild type enzyme. In contrast, the replacement of Arg63 did not cause a significant alteration of the kinetic parameters. The results are in agreement with a previously postulated distant relationship between acid phosphatases, phosphoglycerate mutases, and fructose-2,6-bisphosphatase. These and earlier results are also consistent with the conclusion that 2 histidine residues participate in the catalytic mechanism of acid phosphatases, with His17 playing the role of a nucleophilic acceptor of the phospho group, whereas His303 may act as a proton donor to the alcohol or phenol.

Acyltransferase activities of the high-molecular-mass essential penicillin-binding proteins

The high-molecular-mass penicillin-binding proteins (HMM-PBPs), present in the cytoplasmic membranes of all eubacteria, are involved in important physiological events such as cell elongation, septation or shape determination. Up to now it has, however, been very difficult or impossible to study the catalytic properties of the HMM-PBPs in vitro. With simple substrates, we could demonstrate that several of these proteins could catalyse the hydrolysis of some thioesters or the transfer of their acyl moiety on the amino group of a suitable acceptor nucleophile. Many of the acyl-donor substrates were hippuric acid or benzoyl-D-alanine derivatives, and their spectroscopic properties enabled a direct monitoring of the enzymic reaction. In their presence, the binding of radioactive penicillin to the PBPs was also inhibited.

The catalytic effect of bovine serum albumin on the ortho rearrangement of the potential ultimate carcinogen, N-(sulfooxy)-2-(acetylamino)fluorene, generated enzymically from N-hydroxy-2-(acetylamino)fluorene and evidence for substrate specificity of the enzymic sulfonation of arylhydroxamic acids

This investigation examines the catalytic effect of bovine serum albumin on the ortho rearrangement of the possible ultimate carcinogen, N-(sulfooxy)-2-(acetylamino)fluorene, generated from N-hydroxy-2-(acetylamino)fluorene by the sulfotransferase(s) in the cytosol of rat liver. With various preparations of cytosol, 55-75% of the substrate, N-hydroxy-2-(acetylamino)-fluorene, was found to rearrange to the nonmutagenic and noncarcinogenic o-(sulfooxy) esters, 1- and 3-(sulfooxy)-2-(acetylamino)fluorene, in the presence of bovine serum albumin, while less than 1% of the substrate rearranged in its absence. In presence of bovine serum albumin the cytosolic reduction of N-(sulfooxy)-2-(acetylamino)fluorene to 2-(acetylamino)fluorene decreased by 60-90% and its solvolytic degradation to 4-hydroxy-2-(acetylamino)fluorene by 80-90%. The covalent interaction of enzymatically generated N-(sulfooxy)-2-(acetylamino)fluorene with the nucleophilic acceptors, N-acetyl-L-methionine and guanosine, was lowered by greater than 90% by addition of bovine serum albumin. These measurements indicated that the albumin-catalyzed ortho rearrangement controls the rates of concurrent metabolic and degradative reactions of N-(sulfooxy)-2-(acetylamino)fluorene. The results are in agreement with previous findings of a catalytic effect of serum albumin on the ortho rearrangement of synthetic N-(sulfooxy)-2-(acetylamino)fluorene. In contrast to its catalytic effect on the formation of o-(sulfooxy) esters from N-(sulfooxy)-2-(acetylamino)fluorene, bovine serum albumin had no effect on the formation of o-(acetylamino)fluorenols. To assess the substrate specificity of bovine serum albumin, its effect on the rearrangement of N-hydroxy-2-(benzoylamino)fluorene, a carcinogenic analogue of N-hydroxy-2-(acetylamino)fluorene, was analyzed under conditions of cytosolic sulfonation.(ABSTRACT TRUNCATED AT 250 WORDS)

Polyethylene Glycol Modified Trypsin Kinetics in Buffer and in Benzene: Dipeptide Synthesis

Trypsin, on chemical modification with 2-[(s`-monomethoxy) polyethyleneglycol]-4,6-dichloro-s-triazine, is soluble in benzene. In buffer, this modification increased the amidolytic and esterolytic molecular activity kcat relative to the native enzyme and decreased strongly the Michaelis constant Km of Nα-benzoyl-L-arginine-p-nitroanilide (Bz-L-Arg-NH-Np), whilst that of Nα-benzoyl-L-arginine ethyl ester (Bz-L-ArgOEt) did not change. The modified enzyme catalyzed the aminolysis of the anilide in benzene with trace amounts of water. Syntheses of dipeptides were enzymatically performed with high yields using Narbenzoyl-L-arginine ethyl ester or Nα-benzoyl-L-lysine methyl ester (Bz-L-LysOMe) as acyl-group donors, and L-leucinamide (L-LeuNH2) as an acceptor nucleophile.

Steric course of the N-methylation in the biosynthesis of ergot alkaloids byClaviceps purpurea

Using the chiral methyl group methodology, the methylation step in the biosynthesis of ergot alkaloids catalyzed by the enzyme AdoMet:dimethylallyltryptophan N-methyltransferase was found to proceed with net inversion of methyl group configuration. The enzyme thus conforms to the majority of methyltransferases studied which mediate a direct SN2 transfer of the methyl group from AdoMet to the acceptor nucleophile in a ternary enzyme substrate complex.

Copper-induced dealkylation studies of biologically N-alkylated porphyrins by fast atom bombardment mass spectrometry

Copper has been reported to promote the demethylation of N-methylporphyrins. Copper-induced dealkylation is observed at room temperature in acetonitrile in the presence of a suitable nucleophilic acceptor molecule, or in chloroform alone. Electronic absorption spectra of N-methylprotoporphyrin IX (in the form of its dimethyl ester) in the presence of copper (with either chloroform alone or acetonitrile-dibutylamine as solvent) exhibited a transient spectrum corresponding to a copper chelate of the N-methylprotoporphyrin followed by ejection of the N-methyl group to afford the copper chelate of protoporphyrin. The use of fast atom bombardment mass spectrometry (FAB-MS) to identify the N-alkyl group of a series of N-substituted porphyrins was investigated. The alkyl group released on reaction with copper ions was trapped by an amine (e.g., dodecylamine). The resulting mixture was analysed by positive-ion FAB-MS using 3-nitrobenzyl alcohol as matrix. Molecular ions (MH +) were detected at m/z 200, 214 and 228, corresponding to [CH 3(CH 2) 11NHCH 3 + H] +, [CH 3(CH 2) 11NHC 2H 5 + H] + and [CH 3(CH 2) 11NHC 3H 7 + H] +, respectively. The analysis was extended to N-alkylprotoporphyrins isolated from liver homogenates of mice treated with the porphyria-inducing drug griseofulvin. Using a combination of UV and copper-induced dealkylation studies in FAB-MS, the porphyria-producing pigment was identified as N-methylprotoporphyrin IX.

Further kinetic and molecular characterization of an extremely heat-stable carboxylesterase from the thermoacidophilic archaebacterium Sulfolobus acidocaldarius

The carboxylesterase (serine esterase, EC 3.1.1.1) from Sulfolobus acidocaldarius was purified 940-fold to homogeneity by an improved purification procedure with a yield of 57%. In the presence of alcohols the enzyme catalyses the transfer of the substrate acyl group to alcohols in parallel to hydrolysis. The results show the existence of an alcohol-binding site and a competitive partitioning of the acyl-enzyme intermediate between water and alcohols. Aniline acts also as a nucleophilic acceptor for the acyl group. On the basis of titration with diethyl p-nitrophenyl phosphate, a number of four active centres is determined for the tetrameric carboxylesterase. The sequence of 20 amino acid residues at the esterase N-terminus and the amino acid composition are reported.

Enzymatic approach to the synthesis of a lysine-containing sweet peptide, N-acetyl-L-phenylalanyl-L-lysine.

A sweet tasting peptide containing the l-Iysine residue, yV-acetyl-l-phenylalanyl-l-lysine, was synthesized from /V-acetyl-l-phenylalanine ethyl ester (Ac-Phe-OEt) as donor and l-lysine esters as acceptor nucleophiles by an α-chymotrypsin catalyzed reaction. It was revealed by HPLC analysis that the reaction proceeded most satisfactorily at pH 9 within 3 min in a reaction system containing 100 mM Ac-Phe-OEt, equimolar esters of lysine and 10 μm of the enzyme, where the product yield based on the donor concentration was 53 % for ethyl, 67 % for w-butyl and 31 % for benzyl esters. The highest reaction yield (75 %) was attained by using a double molar excess of lysine /i-butyl ester. The present results suggest that α-chymotrypsin may become a useful tool for the synthesis of peptides containing basic amino acids.

Chemical evidence in favor of a stabilized oxocarbonium-ion intermediate in the NAD + glycohydrolase-catalyzed reactions

The mechanism of the spontaneous solvolysis (hydrolysis and methanolysis) of the nicotinamide-ribose bond of NAD +, studied under unimolecular rate conditions, was compared to the mechanism of the reactions catalyzed by NAD + glycohydrolase. Experimental arguments—i.e., sensitivity of the observed solvolytic rates to the medium effects, stereochemical course of the methanolysis of α- and β-NAD +, partitioning ratio of the ADP-ribosyl intermediate, and entropy of activation—are presented, suggesting that the nonenzymatic solvolysis of β-NAD + proceeds through an enforced preassociation mechanism in which the acceptor molecules, methanol or (and) water, stabilize the developing ADP-ribosyl oxocarbonium ion generated by the bond cleavage. In aqueous solution this intermediate seems too unstable to exist as a fully developed solvent-equilibrated species. In contrast to the spontaneous solvolysis, the ADP-ribosyl intermediate generated in the active site of NAD + glycohydrolase shows a high selectivity when reacting with competing acceptor nucleophiles. This suggests that the enzyme provides a substantial stabilization of the intermediary ADP-ribosyl oxocarbonium ion. We conclude that catalysis of the nicotinamide-ribose bond cleavage by NAD + glycohydrolase would be due, in part, to the ability of the enzyme to effectively stabilize an oxocarbonium ion-like structure in the transition state.

Peptide synthesis catalyzed by polyethylene glycol-modified chymotrypsin in organic solvents.

Chymotrypsin modified with polyethylene glycol was successfully used for peptide synthesis in organic solvents. The benzene-soluble modified enzyme readily catalyzed both aminolysis of N-benzoyl-L-tyrosine p-nitroanilide and synthesis of N-benzoyl-L-tyrosine butylamide in the presence of trace amounts of water. A quantitative reaction was obtained when either hydrophobic or bulky amides of L- as well as D-amino acids were used as acceptor nucleophiles, while almost no reaction occurred with free amino acids or ester derivatives. The acceptor nucleophile specificity of modified chymotrypsin as a catalyst in the formation of both amide and peptide bonds in organic solvents was quite comparable to that in aqueous solution as well as to that of the leaving group in hydrolysis reactions. By contrast, the substrate specificity of modified chymotrypsin in organic solvents was different from that in water since arginine and lysine esters were found to be as effective as aromatic amino acids to form the acyl-enzyme with subsequent synthesis of a peptide bond.

Chromogenic and fluorigenic substrates for sulfurtransferases

Publisher Summary This chapter discusses the chromogenic and fluorigenic substrates for sulfurtransferases. The enzymes rhodanese (thiosulfate : cyanide sulfurtransferase; thiosulfate sulfurtransferase) and thiosulfate reductase catalyze reactions in which a sulfane sulfur atom is transferred from inorganic thiosulfate anion or other suitable donor substrate to a nucleophilic acceptor substrate, which is typically C N - or a thiolate or sulfinate anion. For the chromogenic substrates, 4-(Dimethylamino)-4'-azobenzene (DAB) sulfinic acid is made by reduction of commercial DAB sulfonyl chloride (Aldrich) with excess inorganic sulfite, following the general method of Kulka. DAB thiosulfonate anion is made by treatment of DAB sulfonyl chloride (Aldrich) with l analytical equivalent of inorganic sulfide, following modifications of the general method originated by Traeger and Linde. For the fluorigenic substrates, the same procedures, outlined above for synthesis of chromogenic substrates, can be used to obtain fluorigenic substrates, with the sole alteration that an equivalent amount of 5-dimethylamino-l-naphthalene- sulfonyl chloride (dansyl chloride) is substituted for the dabsyl chloride used as starting material. Moreover, the same thin-layer chromatographic procedures can be used to characterize the products, with the difference that the materials are located on developed plates by fluorescence under long-wavelength UV illumination rather than by color.

Protein bromination by bromoperoxidase from Penicillus capitatus

Publisher Summary Myeloperoxidase (MPO) and bromoperoxidase (BPO), isolated from crude algal extracts of P. capitatus, provide excellent systems for enzymatic radiolabeling of proteins. MPO and BPO are active at neutral pH. Both MPO and the BPO preparations catalyze bromination mainly at tyrosine residues in canine fibrinogen and human serum albumin (HSA). The brominated canine fibrinogen retains all the clotting activity associated with the native protein and no detectable change in biological activity occurs upon the bromination of HSA. Thus, it appears that these enzymes provide a means by which Br may be rapidly and stably attached to protein molecules without serious deleterious effects. This chapter describes the assay method for BPO. The assay utilizes the loss of absorbance at 278 nm upon the halogenation of 1, l-dimethyl-4-chloro-5,5'-cyclohexanedione [monochlorodimedon (MCD)] to the dihalo derivative. Hydrogen peroxide serves as an oxidant to form a reactive enzyme-bound brominating species that reacts with the nucleophilic acceptor to transfer bromine from the enzyme to the acceptor molecule. The time course for the assay is dependent upon the order of the addition of the substrates.

Reactions of phenylium ions with gaseous hydrocarbons. 1. methane, ethane, and propane

The reaction of free tritiated phenylium ion, generated from nuclear decay of [l,4-T 2]-benzene in the presence of simple gaseous hydrocarbons RH (R = CH 3, C 2H 5, C 3H 7; partial pressure: 10-100 torr), yields predominantly the corresponding tritiated C 6H 5R products. The effects of gaseous nucleophilic acceptors (NuH = NH 3, CH 3OH) on the reaction with CH 4, were also investigated. Phenylium ion confirms its exceedingly high reactivity even toward pure σ- -type substrates, as well as its considerable site selectivity, demonstrated by the distinct preference for the C-H bonds of the substrate. The stability features of the ionic intermediates from addition of phenylium ion with RH have been evaluated, as well as their fragmentation and isomerization mechanisms. The behaviour of phenylium ion toward simple aliphatic hydrocarbons in the gas phase (10-100 torr) is discussed and compared with previous mechanistic hypotheses from ICR mass spectrometric studies, carried out at much lower pressures (10 -5 torr).

The activation of human complement component C5 by a fluid phase C5 convertase.

Complement component C5 is converted to C5a and C5b by the cobra venom factor-dependent C3/C5 convertase CVF,Bb (EC 3.4.21.47). The C5 convertase produces selective proteolytic cleavage of an arginyl-leucine peptide bond at positions 74-75 in the alpha chain of C5. Circular dichroism studies in both the far and near UV regions provide evidence that a conformational change accompanies the C5 activation process. When C5 is activated by CVF,Bb in the presence of complement component C6, the C5b,6 complex is formed. However, when C6 is added after C5 has been converted to C5b, the C5b,6 complex fails to form. Therefore, the activation of C5 results in a transient binding site for C6. Hydrophobic sites are probably exposed upon C5 activation because C5b undergoes aggregation when C5 is converted to C5b in the absence of C6. Transmission electron micrographs of the C5 molecule indicate a multilobal, irregular ultrastructure with estimated dimensions of 104 X 140 X 168 A. Aggregated C5b has the appearance of globular particles with a diameter range of 350-700 A. Although C5 shares a number of features with the third component of complement, including a similar ultrastructure and partial sequence homology, C5 is devoid of the unusual thiol ester linkage found in C3. It is the labile thiol ester that permits covalent attachment between C3 and nucleophilic acceptors. In contrast, interactions between C5 and C6 or C5 and membranes remain noncovalent.

Anwendungen der Phasentransfer‐Katalyse, 23. Notiz über Bromiodcarben und zugehörige Halogenaustausch‐prozesse

Applications of Phase Transfer Catalysis, 23. Note on Bromoiodocarbene and on Pertinent Halogen Exchange ProcessesA rapid halogen exchange between HCBr3, HCBr2I, HCBrI2, and HCl3 occurred in the presence of conc. sodium hydroxide and a quaternary ammonium salt. Trapping experiments with various alkenes give cyclopropane derivatives only in 3 cases. Depending on the nucleophilicity of the alkene and the haloform, different proportions of CBr2, CBrI, and Cl2 adducts were obtained. The most nucleophilic acceptor trapped those carbenes preferentially that were formed by elimination of iodide.

Halogenocyclopropanation des éthoxy-alcènes diversement alkylés. Effet syn-directeur des groupes éthoxy et alkyles

Weakly nucleophilic acceptors such as enol ethers can facilitate cycloaddition reactions with monohalocarbenoids. The high yields and stereoselection towards the more highly hindered adducts are interpreted by complexation of the oxygen atom of the substrate with the metal atom of the carbenoid. [Journal translation]