Acyl Transfer Process Research Articles

Ligand-assisted rate acceleration in transacylation by a yttrium-salen complex. Demonstration of a conceptually new strategy for metal-catalyzed kinetic resolution of alcohols.

[reaction: see text]. Yttrium-salen complexes effect transacylation between enolesters and chiral secondary alcohols, resulting in varying degrees of kinetic resolution. Even though the enantioselectivity remains modest (k(fast)/k(slow) up to 4.81), these results represent the first demonstration of a conceptually new metal-catalyzed acyl transfer process that results in kinetic resolution. On the basis of the solid-state structure of the catalyst, a novel associative mechanistic pathway is proposed for the reaction.

Synergistic Effect in the Catalysis by Pyridine N-Oxide-Triethylamine Mixture of Acyl Transfer Processes with Participation of Benzoyl, Diethoxyphosphinoyl, and p-Toluenesulfonyl Chlorides

The kinetics of benzoylation of substituted phenols with benzoyl chloride in the presence of pyridine N-oxide-triethylamine was analyzed with regard to previously reported data on benzoylation, phosphorylation, and sulfonylation of phenols and carboxylic acids. A common mechanism of the synergistic effect was established. The synergistic effect decreases in the series PhCOCl >> (EtO)2POCl > TsCl.

Site-directed mutagenesis and functional analysis of the active-site residues of the E2 component of bovine branched-chain alpha-keto acid dehydrogenase complex.

The catalytic domain of dihydrolipoamide transacylase (E2c) of bovine branched-chain alpha-keto acid dehydrogenase complex (BCKAD) was overexpressed in Escherichia coli. The E2c catalyzes a reversible acyl transfer reaction between acyl-CoA and dihydrolipoamide, which also occurs spontaneously with a much slower rate. The benzene extracts of both the enzyme-catalyzed and the spontaneous reactions mixture have identical ultraviolet absorbance spectra with a maximum at 233-234 nm, which is characteristic of S-acyldihydrolipoamide. The spontaneous reaction rate of various acyl-CoA is in the order of acetoacetyl-CoA > acetyl-CoA > isobutyryl-CoA > isovaleryl-CoA. In other words, the spontaneous acyl transfer is faster when the substituent (R) of acyl-CoA (R-CO-S-CoA) is a more electron-withdrawing group. This result indicates that a negative charge occurs in the substrate during the acyl transfer process. The function of the active-site histidine (His391) and serine (Ser338) of bovine E2c was analyzed by site-directed mutagenesis. Substitution of His391 or Ser338 with alanine caused drastic decreases in catalytic efficiencies by 3-4 orders of magnitude. The residual activity of H391A increased as the pH of the reaction buffer was elevated. These data support the base-catalyzed mechanism inferred from that of chloramphenicol acetyltransferase (CAT). In this reaction, the active-site histidine acts as a general base, and the active-site serine provides a hydrogen bond to the putative negatively charged tetrahedral transition state. Moreover, when Ala348 was changed to valine, the catalytic efficiency for isovaleryl-CoA decreased about 10-fold, and that for acetyl-CoA increased about 3-fold.(ABSTRACT TRUNCATED AT 250 WORDS)

Characterization of the S′‐subsite specificity of bovine pancreatic α‐chymotrypsin via acyl transfer to added nucleophiles

The S'-subsite specificity of bovine pancreatic alpha-chymotrypsin was investigated by acyl transfer reactions using a series of amino-acid- and peptide-derived nucleophiles. The nucleophilic efficiency covers a range of more than three orders of magnitude, reflecting the specificity of the acyl transfer process. Positively charged H-Arg-NH2 was the most efficient nucleophile of the series while peptides with free carboxyl groups show poor nucleophilic behaviour. This is explained by electrostatic interactions with the residues Asp35 and Asp64 of the enzyme. These negatively charged groups, which are localized near the appropriate S' binding sites, repel carboxylate groups of the nucleophiles. There is a good correlation between the nucleophile efficiencies found for different acyl enzymes. An investigation of a series of 14 water-soluble acyl donor esters, differing both in the P1 residue and in the number of amino acids, revealed that the nature of the acyl group affected the acyl-enzyme partitioning between water and added nucleophile in the range of one order of magnitude.

Ionic strength and pH effects in the kinetically controlled synthesis of benzylpenicillin by nucleophilic deacylation of pree and immobilized phenyl-acetyl-penicillin amidase with 6-aminopenicillanic acid

The synthesis of benzylpenicillin (BP) after mixing phenyl-acetyl-glycine(PAG), 6-aminopenicillanic acid (6-APA) and free or immobilized penicillin amidase (E.C.3.5.1.11.) was studied as a function of pH and ionic strength. Before the final equilibrium was reached a kinetically controlled synthesis of BP was observed. Then a transient maximum concentration in BP much larger than the final equilibrium content was synthesized in the acyl-transfer process. The factors influencing this maximum have been analyzed. Increasing ionic strength markedly decreased the maximum in BP and the rate of deacylation of phenyl-acetyl-penicillin amidase by 6-APA. The change was largest when the enzyme was immobilized in a positively charged support, where at low ionic strength the concentration of 6-APA around the enzyme is larger than the bulk concentration due to the partitioning of charged solutes.

Macrocyclic Enzyme Model Systems. Catalytic Activity of Cyclic Peptides Involving Hydrophobic Segments

Abstract In order to develop an effective hydrolase model, BCP-1 was synthesized and its kinetic behavior investigated. The corresponding linear hexapeptide was prepared by the solid phase method and subjected to cyclization by the azide method. BCP-1 enhanced the hydrolysis of p-nitrophenyl carboxylates bearing a long alkyl chain. The pH-rate profile for the hydrolysis of p-nitrophenyl hexadecanoate (PNPP) as effected by BCP-1 was not of a bell-shaped type but a sigmoid, from which the kinetic pKa value was estimated as 12.3. The initial reaction rate was found to level off beyond a certain range of substrate concentration (saturation-type kinetics), the kinetic data being analyzed on the basis of Michaelis-Menten scheme. The large hydrophobic binding ability of BCP-1 toward PNPP (Km(app) \simeq10−6M) was attributed to its double-layered bicyclic structure. The Hammett plot for the hydrolysis of substituted phenyl hexadecanoates gave a satisfactory linear correlation. Consequently, the acyl transfer from the bound substrate to the imidazole anion of the histidyl residue of BCP-1 was referred to the rate-determining step. The large negative entropy change for the acyl transfer step seems to indicate that the Michaelis complex does not have a conformation favorable for such acyl transfer process. The catalytic effectiveness (kcat⁄Km(app)) for the BCP-1 catalysis in PNPP hydrolysis is comparable to that for enzymatic hydrolysis.

Rate acceleration by steric inhibition to solvation of sulphur nucleophiles in acyl transfer processes. A comparison of steric effect in nucleophilic substitutions at carbonyl and aromatic carbon atoms

On increasing the size of the alkyl group in the ortho-position of benzenethiol from methyl to t-butyl the nucleophilicity of benzenethiolate is found to increase in carbonyl carbon atom substitution and to decrease in nucleophilic aromatic substitution; steric inhibition to solvation of the anionic sulphur nucleophile is invoked to account for the rate-accelerting steric effect.

A Macrocyclic Enzyme Model System. Substrate Specificity for the Inclusion into Paracyclophane Oxime and the Subsequent Acyl Transfer Reaction

Abstract In order to characterize the intrinsic nature of 10-hydroxy-11-hydroxyimino[20]paracyclophane (Oxime-I) as being an enzyme model, the acylation reactions of Oxime-I with p-nitrophenyl carboxylates have been investigated in an alkaline aqueous acetone. The usual saturation-type kinetics has been observed for the reactions of p-nitrophenyl decanoate (PNPD), laurate (PNPL), and palmitate (PNPP) with Oxime-I. The acyl transfer reaction was consistent with the following two sequences: (la) pre-equilibrium formation of the inclusion complex between Oxime-I and a substrate, (lb) pre-equilibrium acid dissociation of the inclusion complex, and (lc) rate-determining acyl transfer from a substrate to Oximate-I in the inclusion state; and (2a) pre-equilibrium acid dissociation of Oxime-I, (2b) pre-equilibrium formation of the inclusion complex between Oximate-I and a substrate, and (2c) rate-determining acyl transfer from a substrate to Oximate-I in the inclusion state. On the basis of the compensation correlation between enthalpy and entropy for binding process, the inclusion of a long chain carboxylate into the cavity of Oxime-I is controlled by entropy. It is interesting to note that the binding constants for the present inclusion complexes are considerably larger (K\simeq104–105 M−1) than those for the complexes formed between phenyl esters or azo dyes and cycloamyloses (K\simeq102–103 M−1). Thus, the paracyclophane skeleton provides an effective binding site due to the hydrophobic cavity. Since the most stable inclusion complex was formed with PNPD, the inclusion of the present esters may be not exclusively due to the hydrophobicity provided by an apolar hydrocarbon tail, but significantly due to the molecular geometry or bulkiness characterized by the folded ester molecule. This effect approves the presence of substrate specificity due to the molecular geometry for Oxime-I. In reference to the activation parameters, the acyl transfer process is controlled by entropy in a manner similar to the alkaline hydrolysis in the bulk phase and the complex formation as well. PNPP is least stabilized in the binding process due to the most unfavorable entropy effect, whereas most favorably deacylated in the acyl transfer due to the entropy factor. Consequently, the net change in the free energy is largely reflected on the entropy term. The deceleration effect observed in the reaction of PNPD with Oxime-I at a higher acetone content and the kinetic behavior of alkaline hydrolysis of the three carboxylate esters have also been discussed.