Acylation Rate Research Articles

Modulation of effector affinity by hinge region mutations also modulates switching activity in an engineered allosteric TEM1 β-lactamase switch

RG13 is an engineered allosteric β-lactamase (BLA) for which maltose is a positive effector. RG13 is a hybrid protein between TEM1 BLA and maltose-binding protein (MBP). Maltose binding to MBP is known to convert the open form of the protein to the closed form through conformational changes about the hinge region. We have constructed and genetically selected several variants of RG13 modified in the hinge region of the MBP domain and explored their effect on β-lactam hydrolysis, maltose affinity and maltose-induced switching. Hinge mutations that increased maltose affinity the most (and thus presumably close the apo-MBP domain the most) also abrogated switching the most. We provide evidence for a model of RG13 switching in which there exists a threshold conformation between the open to closed form of the MBP domain that divides states that catalyze β-lactam hydrolysis with different relative rates of acylation and deacylation.

Different Transition-State Structures for the Reactions of β-Lactams and Analogous β-Sultams with Serine β-Lactamases

Beta-sultams are the sulfonyl analogues of beta-lactams, and N-acyl beta-sultams are novel inactivators of the class C beta-lactamase of Enterobacter cloacae P99. They sulfonylate the active site serine residue to form a sulfonate ester which subsequently undergoes C-O bond fission and formation of a dehydroalanine residue by elimination of the sulfonate anion as shown by electrospray ionization mass spectroscopy. The analogous N-acyl beta-lactams are substrates for beta-lactamase and undergo enzyme-catalyzed hydrolysis presumably by the normal acylation-deacylation process. The rates of acylation of the enzyme by the beta-lactams, measured by the second-order rate constant for hydrolysis, kcat/K(m), and those of sulfonylation by the beta-sultams, measured by the second-order rate constant for inactivation, k(i), both show a similar pH dependence to that exhibited by the beta-lactamase-catalyzed hydrolysis of beta-lactam antibiotics. Electron-withdrawing groups in the aryl residue of the leaving group of N-aroyl beta-lactams increase the rate of alkaline hydrolysis and give a Bronsted beta(lg) of -0.55, indicative of a late transition state for rate-limiting formation of the tetrahedral intermediate. Interestingly, the corresponding Bronsted beta(lg) for the beta-lactamase-catalyzed hydrolysis of the same substrates is -0.06, indicative of an earlier transition state for the enzyme-catalyzed reaction. By contrast, although the Bronsted beta(lg) for the alkaline hydrolysis of N-aroyl beta-sultams is -0.73, similar to that for the beta-lactams, that for the sulfonylation of beta-lactamase by these compounds is -1.46, compatible with significant amide anion expulsion/S-N fission in the transition state. In this case, the enzyme reaction displays a later transition state compared with hydroxide-ion-catalyzed hydrolysis of the beta-sultam.

Inhibition of Class D β-Lactamases by Diaroyl Phosphates

The production of beta-lactamases is an important component of bacterial resistance to beta-lactam antibiotics. These enzymes catalyze the hydrolytic destruction of beta-lactams. The class D serine beta-lactamases have, in recent years, been expanding in sequence space and substrate spectrum under the challenge of currently dispensed beta-lactams. Further, the beta-lactamase inhibitors now employed in medicine are not generally effective against class D enzymes. In this paper, we show that diaroyl phosphates are very effective inhibitory substrates of these enzymes. Reaction of the OXA-1 beta-lactamase, a typical class D enzyme, with diaroyl phosphates involves acylation of the active site with departure of an aroyl phosphate leaving group. The interaction of the latter with polar active-site residues is most likely responsible for the general reactivity of these molecules with the enzyme. The rate of acylation of the OXA-1 beta-lactamase by diaroyl phosphates is not greatly affected by the electronic effects of substituents, probably because of compensation phenomena, but is greatly enhanced by hydrophobic substituents; the second-order rate constant for acylation of the OXA-1 beta-lactamase by bis(4-phenylbenzoyl) phosphate, for example, is 1.1 x 10(7) s(-)(1) M(-)(1). This acylation reactivity correlates with the hydrophobic nature of the beta-lactam side-chain binding site of class D beta-lactamases. Deacylation of the enzyme is slow, e.g., 1.24 x 10(-)(3) s(-)(1) for the above-mentioned phosphate and directly influenced by the electronic effects of substituents. The effective steady-state inhibition constants, K(i), are nanomolar, e.g., 0.11 nM for the above-mentioned phosphate. The diaroyl phosphates, which have now been shown to be inhibitory substrates of all serine beta-lactamases, represent an intriguing new platform for the design of beta-lactamase inhibitors.

Efficient Synthesis of Tyrosine-Derived Marine Sponge Metabolites via Acylation of Amines with a Coumarin

Condensation of N-acetylglycine with aldehyde 15 in acetic anhydride gave acetamido coumarin 16. Hydrolysis to the enol coumarin 17 and reaction with hydroxylamine gave the oximino coumarin 18. Reaction of the oximino coumarin 18 with a range of nucleophiles gave the phenolic oximes in excellent yield. The rates of acylation of histamine with the oximino coumarin 18 and methyl ester 9 were compared. Oxidative spirocyclization of three representative phenolic oximes with polymer-supported (diacetoxyiodo) benzene gave the spiroisoxazolines.

Hirudin Binding Reveals Key Determinants of Thrombin Allostery

Thrombin exists in two allosteric forms, slow (S) and fast (F), that recognize natural substrates and inhibitors with significantly different affinities. Because under physiologic conditions the two forms are almost equally populated, investigation of thrombin function must address the contribution from the S and F forms and the molecular origin of their differential recognition of ligands. Using a panel of 79 Ala mutants, we have mapped for the first time the epitopes of thrombin recognizing a macromolecular ligand, hirudin, in the S and F forms. Hirudin binding is a relevant model for the interaction of thrombin with fibrinogen and PAR1 and is likewise influenced by the allosteric S-->F transition. The epitopes are nearly identical and encompass two hot spots, one in exosite I and the other in the Na+ site at the opposite end of the protein. The higher affinity of the F form is due to the preferential interaction of hirudin with Lys-36, Leu-65, Thr-74, and Arg-75 in exosite I; Gly-193 in the oxyanion hole; and Asp-221 and Asp-222 in the Na+ site. Remarkably, no correlation is found between the energetic and structural involvements of thrombin residues in hirudin recognition, which invites caution in the analysis of protein-protein interactions in general.

Highly Enantioselective Acylation of rac‐Alkyl Lactates Using Candida antarctica Lipase B.

AbstractFor Abstract see ChemInform Abstract in Full Text.

Structure-Activity Relationships of Different β-Lactam Antibiotics against a Soluble Form of Enterococcus faecium PBP5, a Type II Bacterial Transpeptidase

Penicillin-binding proteins (PBPs) catalyze the essential reactions in the biosynthesis of cell wall peptidoglycan from glycopeptide precursors. beta-Lactam antibiotics normally interfere with this process by reacting covalently with the active site serine to form a stable acyl-enzyme. The design of novel beta-lactams active against penicillin-susceptible and penicillin-resistant organisms will require a better understanding of the molecular details of this reaction. To that end, we compared the affinities of different beta-lactam antibiotics to a modified soluble form of a resistant Enterococcus faecium PBP5 (Delta1-36 rPBP5). The soluble protein, Delta1-36 rPBP5, was expressed in Escherichia coli and purified, and the NH(2)-terminal protein sequence was verified by amino acid sequencing. Using beta-lactams with different R1 side chains, we show that azlocillin has greater affinity for Delta1-36 rPBP5 than piperacillin and ampicillin (apparent K(i) = 7 +/- 0.3 microM, compared to 36 +/- 3 and 51 +/- 10 microM, respectively). Azlocillin also exhibits the most rapid acylation rate (apparent k(2) = 15 +/- 4 M(-1) s(-1)). Meropenem demonstrates an affinity for Delta1-36 rPBP5 comparable to that of ampicillin (apparent K(i) = 51 +/- 15 microM) but is slower at acylating (apparent k(2) = 0.14 +/- 0.02 M(-1) s(-1)). This characterization defines important structure-activity relationships for this clinically relevant type II transpeptidase, shows that the rate of formation of the acyl-enzyme is an essential factor determining the efficacy of a beta-lactam, and suggests that the specific side chain interactions of beta-lactams could be modified to improve inactivation of resistant PBPs.

A mathematical model for mammary fatty acid synthesis and triglyceride assembly: the role of stearoyl CoA desaturase (SCD)

An increase in the proportion of unsaturated fatty acids in milk is considered desirable for human health. A prerequisite for the manipulation of milk fat composition is a co-ordinated understanding of the complex interactions in its biosynthesis. It has been suggested that an increase in the expression of mammary stearoyl-CoA-desaturase (SCD) would enrich mono-unsaturated fatty acids in milk, and therefore improve its nutritional properties. To investigate the potential effects of changes in expression of mammary enzymes and substrate availability on milk fat composition, we constructed, parameterized and evaluated a mechanistic mathematical model of fatty acid biosynthesis and milk-fat triglyceride assembly. The objective was to describe changes in the amount and composition of milk fat produced by bovine mammary cells due to changes in nutrition. Using the model we found that a 50% up-regulation in SCD activity increased the molar fraction of milk triglyceride 18:1 from 0.30 to 0.33 and 16:1 from 0.04 to 0.06. Up-regulation of SCD therefore did not appear to be the optimal method for increasing the content of unsaturated fatty acids in milk fat. The model was also used to determine the likely rate-limiting processes for the incorporation of unsaturated fatty acids into milk fat. Halving the concentration of glycerol 3-phosphate increased the molar fraction of milk triglyceride 18:1 from 0.30 to 0.35 and decreased the molar fraction of milk triglyceride 16:0 from 0.30 to 0.22. This achieved the desirable outcome of producing more unsaturated low-fat milk. Our model also predicted that a K232A mutation in the bovine mammary DGAT1 gene that is linked with an increase in milk fat yield would be consistent with a 120% increase in the DGAT acylation rate and also would be associated with a decrease in milk mono-unsaturated fatty acids.

Highly Enantioselective Acylation of rac-Alkyl Lactates Using Candida antarctica Lipase B

By using Candida antarctica lipase B under mild conditions, the highly enantioselective acylation of alkyl (R)-lactate from racemic mixture with vinyl alkanoate has been accomplished. In this research effects of the organic solvent, the alkyl chain length of the alkyl lactates and of the vinyl alkanoates, and the reaction temperature on the enantiomeric excess as well as the reaction rate, were investigated. In all cases, only alkyl (R)-lactate was stereoselectively acylated at >99.5% ee. The lipase-catalyzed acylation rate of the alkyl lactates was affected by the nature of the organic solvents, but showed no correlation to log P of the solvent. The lipase-catalyzed acylation rate of the alkyl lactates was enhanced by increasing the chain length of the vinyl alkanoate from acetyl to butanoyl and by raising the reaction temperature to 65 °C. Finally, the lipase-catalyzed acylation and subsequent vacuum distillation successfully provided both butyl (R)-O-butanoyllactate and butyl (S)-lactate in excellent yields (48%) and enantioselectivities (>99.5% ee) on a large scale. It is expected that the present method will prove to be more efficient in achieving the chiral resolution of racemic alkyl lactate than other conventional methods in terms of environmental friendliness and simplicity.

On the role of zeolite structure and acidity in toluene acylation with isobutyric acid derivatives

Zeolite catalysts of various structures and type and concentration of acid sites were investigated in a liquid phase acylation of toluene with derivatives of isobutyric acid. The highest conversions of isobutyryl chloride or isobutyric anhydride were obtained over three-dimensional large-pore zeolite Beta and zeolite Y possessing medium concentration of acid sites. In general, the conversion of acylating agents increases with increasing pore size diameter and dimensionality of the channel system. Large-pore zeolites with three-dimensional channel system enable easier desorption and transport of bulky isopropyltolyl ketone. The activity of the zeolites used can be also controlled by tailoring their hydrophobic/hydrophilic properties. The higher initial rate of toluene acylation with isobutyric anhydride compared to isobutyryl chloride was found. However, only one possible acylating group of isobutyric anhydride is used for acylation reaction.

Regioselective acylation of flavonoids catalyzed by immobilized Candida antarctica lipase under reduced pressure.

A single-step acylation of rutin and naringin, catalyzed by immobilized Candida antarctica lipase B in 2-methyl-2-butanol, occurred preferentially on the primary hydroxyl group. Using palmitic methyl ester as acyl donor, the acylation rate of naringin was 10-fold higher than that of rutin. Under optimal conditions, i.e. a molar ratio acyl donor/naringin of 7:1 and 200 mbar, 92% naringin was acylated.

Effect of acyl donor chain length and substitutions pattern on the enzymatic acylation of flavonoids

Rutin and esculin were enzymatically acylated with different aliphatic acids as acyl donors (fatty acids, dicarboxylic acids and ω-substituted fatty acids) by an immobilized lipase from Candida antarctica. The effect of the water content and the acyl donors pattern on the flavonoid initial acylation rate and conversion yield were investigated. The obtained results indicated that the water content of the medium has a strong effect on the performance of these reactions. The best conversion yields were reached when the water content was kept lower than 200 ppm. At low water content of the medium, these syntheses are influenced by carbon chain length and substitution pattern of the acyl donors. Higher conversion yields of esculin and rutin (>70%) were obtained with aliphatic acids having high carbon chain length (>12). Moreover, it has been found that the amine and thiol groups on ω-substituted fatty acid chain were unfavourable to these reactions. The 1 H NMR and 13 C NMR analyses of some synthesized esters (esculin and rutin palmitate) show that only monoesters were produced and that the esterification takes place on the primary OH of glucose moiety of the esculin and on the secondary 4′′′-OH of the rhamnose residue of rutin.

Selective esterifications of alcohols and phenols through carbodiimide couplings.

Esterification of carboxylic acids capable of forming ketene intermediates upon treatment with carbodiimides permits the selective acylation of alcohols in the presence of phenols lacking strong electron-withdrawing groups. The selectivity of acylations involving highly acidic phenols could be reversed through the addition of catalytic amount of acid. Esterification of other carboxylic acids was found to proceed through the formation of symmetric anhydrides and provide the opposite chemoselectivity. In both cases the relative acylation rates of substituted phenols are consistent with a reaction mechanism involving an attack of phenolate anions on electrophilic intermediates such as ketenes and symmetric anhydrides, with the carbodiimides serving both as an activating reagent and as a basic catalyst.

Solid/gas biocatalysis: an appropriate tool to study the influence of organic components on kinetics of lipase-catalyzed alcoholysis

The influence of the addition of an extra component in a gaseous reaction medium, on the kinetics of alcoholysis of methyl propionate and n-propanol catalyzed by immobilized lipase B from Candida antarctica was studied in a continuous solid/gas reactor. In this reactor, the solid phase is composed of a packed enzymatic sample, which is percolated by gaseous nitrogen, simultaneously carrying gaseous substrates and additional components to the enzyme while removing reaction products. The system permits to set thermodynamic activity of all gaseous components (substrates or not) independently at the desired values. This allows in particular to study the influence of an extra added component at a constant thermodynamic activity value, contrary to classical solid/liquid system, which involves large variations of thermodynamic activity of added solvent, when performing full kinetic studies. Alcohol inhibition constant ( K I) and methyl propionate and propanol dissociation constants ( K MP and K P) have been determined in the solid/gas reactor in the presence of 2-methyl-2-butanol, and compared with values previously obtained in the absence of added component and in the presence of water. Complementary experiments were carried out in the presence of an apolar compound (hexane) and led to the conclusion that the effect of added organic component on lipase-catalyzed alcoholysis is related to their competitive inhibitory character towards first substrate methyl propionate. The comparison of data obtained in liquid or with gaseous 2-methyl-2-butanol shows that lower K MP and K I are found in gaseous medium, which would correspond on the one hand to a lower acylation rate k 2, and on the other hand to a higher binding rate k 1 between substrate and free enzyme in gaseous medium.

Selective enzymatic synthesis of amide surfactants from diethanolamine

Kinetic strategies for selective preparation of amides from diethanolamine have been designed using Novozym 435 lipase as a biocatalyst. Two different routes, direct acylation and transacylation, have been optimized. In n-hexane, the reaction is more selective for formation of the amide by direct acylation, while in dioxane, the O-acylation reaction is highly favored. However, the initial rates of direct acylation and transacylation are both higher in dioxane than in n-hexane because of the low solubilities of both diethanolamine and its corresponding ion-pair complex with the fatty acid. At 30 °C, the high viscosities of mixtures of diethanolamine with fatty acids limit the extent of reaction and the corresponding yields of the amide. This effect is greater in n-hexane in which the viscosity (5.52 cSt) was four times greater than dioxane. An increase in temperature to 60 °C increases the conversion and decreases the viscosity of the n-hexane solution to 0.8–0.9 cSt. At 60 °C when equimolar amounts of reactants are employed, the transacylation route produces both higher conversions (71–77 mol%) and greater selectivities to the amide (74–94%) than the direct acylation reaction (69–74 mol% conversion and 76–86% selectivity). For both synthesis routes, the volumetric productivity of the reactor is restricted for reactant concentrations above 0.8 M. At 60 °C, conversions via the direct acylation reaction can be increased to 92 and 80 mol% in dioxane and n-hexane, respectively. These conversions require using a two-fold excess of diethanolamine. The resultant selectivities are 98 and 100%, respectively.

Arrhenius analysis of the electrophorus electricus acetylcholinesterase-catalyzed hydrolysis of acetylthiocholine.

Ellman's method was used to determine the Michaelis-Menten parameters for the hydrolysis of acetylthiocholine by Electrophorus electricus acetylcholinesterase from 12 to 37 degrees C. Arrhenius analysis revealed that the activation energy for formation of the enzyme/substrate complex is 22.2 +/- 1.1 kJ/mole. The Arrhenius plot of k(cat) is markedly curved and attributed to comparable rates of acylation and deacylation due to the absence of evidence for a temperature-dependent enzyme conformational change by differential scanning calorimetry.

The kinetic properties of the carboxy terminal domain of the Bacillus licheniformis 749/I BlaR penicillin-receptor shed a new light on the derepression of beta-lactamase synthesis.

To study the properties of the BlaR penicillin-receptor involved in the induction of the Bacillus licheniformisbeta-lactamase, the water-soluble carboxy terminal domain of the protein (BlaR-CTD) was overproduced in the periplasm of Escherichia coli JM105 and purified to protein homogeneity. Its interactions with various beta-lactam antibiotics were studied. The second-order acylation rate constants k2/K' ranged from 0.0017 to more than 1 micro M-1s-1 and the deacylation rate constants were lower than 4 x 10-5 s-1. These values imply a rapid to very rapid formation of a stable acylated adduct. BlaR-CTD is thus one of the most sensitive penicillin-binding proteins presently described. In the light of these results, the kinetics of beta-lactamase induction in Bacillus licheniformis were re-examined. When starting with a rather high cell density, a good beta-lactamase substrate such as benzylpenicillin is too sensitive to beta-lactamase-mediated hydrolysis to allow full induction. By contrast, a poor beta-lactamase substrate (7-aminocephalosporanic acid) can fully derepress beta-lactamase expression under conditions where interference of the antibiotic with cell growth is observed. These results suggest that acylation of the penicillin receptor is a necessary, but not sufficient, condition for full induction.

Alcoholysis catalyzed by Candida antarctica lipase B in a gas/solid system: effects of water on kinetic parameters

The influence of water on the kinetics of alcoholysis of methyl propionate and n-propanol catalyzed by immobilized lipase B from Candida antarctica was studied in a continuous solid/gas reactor. In this reactor, the solid phase is composed of a packed enzymatic sample which is percolated by gaseous nitrogen, simultaneously carrying gaseous substrates to the enzyme while removing reaction products. In this system, interactions between the enzyme and nonreacting molecules are avoided, since no solvent is present, and it is thus more easy to assess the role of water. To this end, alcohol inhibition constant, substrates dissociation constants as well as acylation rate constant and ratio of acylation to deacylation rate constants have been determined as a function of water activity ( a w). Data obtained highlight that n-propanol inhibition constant and dissociation constant of methyl propionate are a lot affected by a w variations whereas water has no significant effect on the catalytic acylation step nor on the ratio of acylation to deacylation rate constants. These results suggest the water-independent character of the transition step.

Different Acylating Agents in the Synthesis of Aromatic Ketones on Sulfated Zirconia

Sulfated zirconia (SZ) has been used as a heterogeneous catalyst in the synthesis of aromatic ketones. A number of carboxylic anhydrides and acid chlorides proved to be appropriate acylating agents in the reaction with anisole and chlorobenzene. The rate of anisole acylation was found to be dependent on the type of acylating agent used. Thus, acetic anhydride and benzoic anhydride reacted faster on SZ with anisole than benzoyl chloride. Successful SZ-catalyzed acylations of chlorobenzene were limited to acceptor substituted benzoyl chlorides as acylating agents. Acetic anhydride and benzoic anhydride did not react with chlorobenzene in the presence of SZ.

Lipase-catalyzed esterification of rutin and naringin with fatty acids of medium carbon chain

Flavonoids rutin and naringin were acylated with fatty acids of medium carbon chain (with 8–12 carbon atoms on their molecule) in a reaction catalyzed by immobilized lipase from Candida antarctica (Novozyme) in various solvent systems. The reaction parameters affecting the acylation rate and the conversion of the enzymatic process, such as the nature of the organic solvent and acyl donor used, the water activity ( a w) of the system, as well as the kinetic of the reaction have been investigated. In all cases studied, only flavonoid monoester is identified as the product, which indicates that this lipase-catalyzed esterification is regioselective. The enzymatic acylation of flavonoids seems to follow Michaelis–Menten kinetics.