Nitrophenyl Esters Research Articles

Molecular recognition by natural macrocycles. Part II. Esterolytic activity and chiral discrimination of amino acid derivatives by the zwitterionic form of (+)-tubocurarine †

The esterase and complexation properties of the zwitterionic form of a macrocyclic alkaloid (+)-tubocurarine possessing two phenolic nucleophilic groups are described. Cleavage of 4-nitrophenyl esters of N-protected phenylalanine enantiomers involves reaction paths with one and two alkaloid molecules. Substrate binding enantioselectivity is large and is opposite to the kinetic enantioselectivity, leading to the modest observed enantioselectivity of the reaction, which reaches a maximum (kL/kD ≈ 1.5) at ca. 0.002 mol dm–3 alkaloid concentration and practically disappears on going to 0.01 mol dm–3 alkaloid solution. Addition of boric acid initially enhances the reaction rate and enantiospecificity, but in more concentrated borate solutions the expected inhibition due to blocking of the phenolate groups is observed. For the first time reported for an alkaloid, the esterolytic activity of (+)-tubocurarine towards 4-nitrophenyl acetate falls on a common Bronsted plot together with cyclodextrins and some synthetic macrocycles. Binding of enantiomers of differently charged derivatives of alanine, phenylalanine and β-phenylethylamine to the zwitterionic form of (+)-tubocurarine in aqueous solution was studied by 1H NMR and fluorescence titration. The binding constants vary from <5 dm3 mol–1 for alanine to ca. 50 dm3 mol–1 for phenylalanine derivatives and the binding enantioselectivity varies from marginal for N-acetylphenylalanine enantiomers to a quite notable, three-fold differentiation between L- and D-phenylalanine. While the enantiospecificity depends primarily on electrostatic interactions, the overall stability is determined by guest hydrophobicity. This conclusion was confirmed by docking calculations for enantiomers of phenylalanine. Addition of amino acid derivatives to solutions containing (+)-tubocurarine and highly fluorescent 8-anilinonaphthalenesulfonate anion leads to enantioselective spectral responses which are indicative of formation of ternary complexes.

Kinetics and mechanism of phosphorylation of monoethanolamine in reversed micelles of a cationic surfactant

The reactions of monoethanolamine with 4-nitrophenyl esters of tetracoordinated phosphorus acids in chloroform in the presence of 2-hydroxyethyldimethylpentadecylammonium bromide were studied. The effective rate constants of the reactions are increased by more than two orders of magnitude in the presence of the micelle-forming cationic surfactant. The latter also favors the formation of the product ofO-phosphorylation of monoethanolamine and leads to the appearance of a new ionic form of the 4-nitrophenol-monoethanolamine complex.

Aminolysis of 4-nitrophenyl esters of phosphorus acids in reversed micelles of 2-hydroxyethyldimethylpentadecylammonium bromide

The reactions ofn-cetyl- andn-hexylamines with 4-nitrophenyl esters of tetracoordinated phosphorus acids in chloroform in the presence of 2-hydroxyethyldimethylpentadecylammonium bromide and the influence of the latter on the acid—base equilibrium of the bromphenol blue dye (BPB) were studied by the spectrophotometric method. In the presence of reversed micelles of the cationic surfactant, the observed rate constant of aminolysis increases by more than two orders of magnitude. The catalytic efficiency of the micelles increases as the concentration of the long-chain amine decreases and on going from the latter to a short-chain amine. The acid—base equilibrium of BPB in micellar solutions is shifted due to the formation of a complex between the surfactant and the BPB dianion.

Catalysis of Hydrolysis and Transesterification Reactions of p-Nitrophenyl Esters by a Designed Helix−Loop−Helix Dimer

KO-42, a polypeptide with 42 amino acid residues has been designed to fold into a hairpin helix−loop−helix motif that dimerizes and forms a four-helix bundle. The solution structure of the folded KO-42 dimer has been determined by NMR and CD spectroscopy and ultracentrifugation. On the surface of the folded polypeptide a reactive site has been engineered that is capable of catalyzing acyl-transfer reactions of reactive esters. The reactive site of KO-42 contains six histidine residues with perturbed pKa values. The pKas of His-15, His-30, and His-34 are close to 5, whereas those of His-11, His-19, and His-26 are close to 7, with nonideal titration curves. The second-order rate constant for the KO-42 catalyzed hydrolysis of mono-p-nitrophenyl fumarate at pH 4.1 and 290 K is 0.1 M-1 s-1, which is 1140 times larger than that of the 4-methylimidazole (4-MeIm) catalyzed reaction, 8.8 × 10-5 M-1 s-1. The second-order rate constant for the KO-42 catalyzed transesterification of mono-p-nitrophenyl fumarate to for...

Expression and characterization of recombinant single-chain Fv and Fv fragments derived from a set of catalytic antibodies

The generation of catalytic antibodies should enable the catalysis of reactions for which no enzymatic or chemical catalyst is currently available. In previous studies, we established a series of catalytic antibodies capable of hydrolysing p-nitrobenzyl (pNB) and p-nitrophenyl (pNP) esters. A group of these catalytic antibodies exhibited high reactivity and substrate specificity, yet each individual antibody demonstrated different kinetic parameters. In order to study the molecular basis for these differences, we have cloned, sequenced and expressed the variable regions of this group of antibodies as functional scFv and Fv in bacteria. The variable region of the heavy chain is derived from a novel germline gene of the J558 family whereas the light chain comes from a germline gene previously found in our catalytic antibodies catalysing the hydrolysis of only nitrophenyl esters, demonstrating that the heavy chain determines the specificity for the nitrobenzyl esters. Several different expression systems were examined for their ability to produce catalytically active antibodies. When expressed as an scFv, both refolded and secreted scFvs exhibited catalytic activity although yields of expressed protein were low. The secreted scFvs had higher specific activity. On the other hand, Fv fragments were expressed in sufficient quantities to allow kinetic analysis. Levels of expression were dependent on the sequence of V L used. Using this expression system, the relative contributions of the individual light and heavy chains to catalysis and binding could be evaluated. Both original V H and V L regions are required for hapten binding, although the V H is more crucial for catalysis. By replacing the CDR3 of the heavy chain with a random sequence, it was shown to be essential for both binding and catalysis. This expression system together with site-directed mutagenesis should enable a more detailed study of the catalytic mechanism of this set of antibodies.

Mechanism of inactivation of a catalytic antibody by p-nitrophenyl esters.

Antibody CNJ206 catalyses the hydrolysis of p-nitrophenyl esters with significant rate enhancement; however, after a few cycles, 90% of the catalytic activity of CNJ206 is irreversibly lost. This report investigates the properties of the inactivated Fab (fragment antigen binding). After inactivation, the residual esterase activity of CNJ206 is similar to that of the catalytic antibody inhibited by the transition-state analogue (TSA) used to elicit it; the affinity of CNJ206 for the TSA is also dramatically lowered. Here we propose a simple scheme that accounts for the steady-state kinetics of inactivation. The following lines of evidence, when taken together, suggest that stable acylated tyrosine side chains within or close to the Fab combining site are involved in the inactivation process: isoelectric focusing and matrix-assisted-laser-desorption-ionisation-time-of-flight (MALDI-TOF) mass spectrometry show that incubation with substrate results in several acylated Fab species; inactivation is stable at pH 8, is reversed by mild hydroxylamine treatment and follows the same kinetics as inhibition of binding, which is slowed down by the presence of the TSA hapten. Analysis of the Fab-TSA X-ray structure shows that three tyrosine residues are potential candidates for the inactivation of CNJ206 by its substrates, Tyr L96 being the most likely one; this also suggests that site-directed mutation of one or more of these residues might prevent substrate inactivation and significantly improve catalysis.

Ester Hydrolysis by a Catalytic Cyclodextrin Dimer Enzyme Mimic with a Metallobipyridyl Linking Group

A β-cyclodextrin dimer with a linking bipyridyl group is synthesized as a catalyst precursor, a holoenzyme mimic. It binds both ends of potential substrates into the two different cyclodextrin cavities, holding the substrate ester carbonyl group directly above a metal ion bound to the bipyridyl unit. The result is very effective ester hydrolysis with good turnover catalysis. For example, a Cu(II) complex accelerates the rate of hydrolysis of several nitrophenyl esters by a factor of 104−105, with at least 50 turnovers and no sign of product inhibition. In the best case, with an added nucleophile that also binds to the metal ion, a rate acceleration of 1.45 × 107 over the background reaction rate was observed. Hydrolysis by a catalyst with only one cyclodextrin binding group is significantly slower than in the bidentate binding cases. As expected, the binding of a transition state analogue to these catalysts is stronger with the metal ion present than without. This and kinetic evidence point to a mechanism in which the metal ion plays a bifunctional acid−base role, enforced by the binding geometry that holds the substrate functionality right on top of the catalytic metal ion.

Investigation of two glycosylated forms of bile-salt-dependent lipase in human pancreatic juice.

Pure human pancreatic bile-salt-dependent lipase, devoid of its oncofetal glycoform [Mas, E., Abouakil, N., Roudani, S., Miralles, F., Guy-Crotte., O., Figarella, C., Escribano, M. J. & Lombardo, D. (1993) Biochem. J. 289, 609-615], was analyzed on immobilized concanavalin A (ConA). Two variants were separated: an unabsorbed ConA-unreactive fraction; and an absorbed ConA-reactive fraction. Carbohydrate compositions of ConA-reactive and ConA-unreactive fractions were not significantly different, and analysis of 3H-labelled oligosaccharides liberated from these fractions on the ConA-Sepharose column indicated that the fractionation of the bile-salt-dependent lipase on this column depends upon oligosaccharide structures. The activity of the ConA-reactive fraction was however much lower, independent of the substrate (4-nitrophenyl hexanoate or cholesteryl esters), than that of the ConA-unreactive fraction. Therefore, catalytic constants for the hydrolysis of 4-nitrophenyl hexanoate were determined; both fractions had quite similar Km, while the kcat for the ConA-unreactive fraction was 3-4-fold higher than that of the ConA-reactive fraction. ConA-reactive and ConA-unreactive fractions were shown to have slightly different molecular masses and different amino acid compositions. Cleavage patterns after cyanogen bromide treatment of the ConA-reactive and ConA-unreactive fractions suggested that the ConA-reactive (high Mr form) and ConA-unreactive (low Mr form) forms could be different isoforms of the bile-salt-dependent lipase secreted by the human pancreas.

Nucleophilic displacement at the benzoyl centre: a study of the change in geometry at the carbonyl carbon atom

M. J. Colthurst and A. Williams, J. Chem. Soc., Perkin Trans. 2, 1997, 1493 DOI: 10.1039/A700686A

Effects of cyclodextrin on hydrolysis and the Smiles rearrangement of salicylic acid esters

The effect of β-cyclodextrin (β-CD) on “aqueous” hydrolysis of methyl,p-, andm-nitrophenyl salicylates as well as on the Smiles rearrangement ofp-nitrophenyl salicylate was studied. No effect of β-CD on the pH-independent rate constant of “aqueous” hydrolysis of methyl ester was observed, while β-CD accelerated “aqueous” hydrolysis of nitrophenyl esters byca. 10 times. The inclusion of these esters into the cavity of β-CD is accompanied by a change in the mechanism of hydrolysis: free ester in the deprotonated form undergoes hydrolysis through the mechanism of intramolecular general base catalysis, while the ester bound to cyclodextrin is hydrolyzed due to the nucleophilic attack of the deprotonated hydroxyl group of β-CD at a neutral substrate molecule. The effects of cyclodextrin on the rate constant of borate-catalyzed hydrolysis were interpreted by assuming that the substrate bound to β-CD undergoes borate-assisted attack at the deprotonated cyclodextrin hydroxyl group. The Smiles rearrangement, which is an intramolecular nucleophilic substitution reaction, is accelerated in the presence of β-CD.

Isolation and Characterization of Novel Long-Chain Acyl-CoA Thioesterase/Carboxylesterase Isoenzymes fromCandida rugosa

Long-chain acyl-CoA thioesterases, which catalyze the cleavage of acyl-CoA's to free fatty acids and CoASH, are abundant in animal cells. However, in yeast little is known about presence and function of acyl-CoA thioesterase activity. Therefore a commercial lipase preparation from the yeastCandida rugosawas investigated and found to contain high myristoyl-CoA thioesterase activity. Hydrophobic interaction chromatography separated the activity into three peaks, of which two enzymes (YTE-1 and YTE-2) were purified to apparent homogeneity with molecular masses of about 40 kDa as determined by size-exclusion chromatography and SDS–PAGE. The employed purification protocol resulted in final preparations with specific activities of about 90 μmol/mg/min with myristoyl-CoA as substrate. YTE-1 and YTE-2 showed similar kinetic properties and YTE-1 was characterized in detail. Acyl-CoA chain-length specificity showed that YTE-1 was not active on acyl-CoAs shorter than decanoyl-CoA, at the substrate concentrations tested. The best substrates were C14–C18acyl-CoAs withVmaxvalues of about 150 μmol/mg/min andKmvalues of 15–46 μm. The enzyme was very active with lauroyl-CoA (Vmaxabout 400 μmol/mg/min) although theKmwas high (about 325 μm). The purified enzyme was also active on short-chain nitrophenyl esters but inactive with tributyrin. Treatment of the protein withN-glycosidase F decreased the molecular mass about 1–2 kDa, indicating the presence of carbohydrate of the high mannose type. Diisopropyl fluorophosphate (DFP) inhibited the enzyme activity efficiently and the protein was covalently labeled with [3H]DFP.p-Chloromercuribenzoic acid inhibited the thioesterase activity but did not affect carboxylesterase activity. N-terminal sequence analysis and labeling by DFP suggest that these long-chain acyl-CoA thioesterases belong to a novel group of yeast serine esterases.

Rapid burst kinetics in the hydrolysis of 4-nitrophenyl acetate by penicillin G acylase from Kluyvera citrophila. Effects of mutation F360V on rate constants for acylation and de-acylation

The kinetics of release of 4-nitrophenol were followed by stopped-flow spectrophotometry with two 4-nitrophenyl ester substrates of penicillin G acylase from Kluyvera citrophila. With the ester of acetic acid, but not of propionic acid, there was a pre-steady-state exponential phase, the kinetics of which were inhibited by phenylacetic acid (a product of hydrolysis of specific substrates) to the extent predicted from Ki values. This was interpreted as deriving from rapid formation (73 mM-1.s-1) and slow hydrolysis (0.76 s-1) of an acetyl derivative of the side chain of the catalytic-centre residue Ser-290. With the mutant F360V, which differs from the wild-type enzyme in its ability to hydrolyse adipyl-L-leucine and has a kcat for 4-nitrophenyl acetate one-twentieth that of the wild-type enzyme, the corresponding values for the rates of formation and hydrolysis of the acetyl-enzyme were 11.1 mM-1.s-1 and 0.051 s-1 respectively. The ratio of these rate constants was three times that for the wild-type enzyme, suggesting that the mutant is less impaired in the rate of formation of an acetyl-enzyme than in its subsequent hydrolysis.

Transfer of a positively charged acyl group between substituted phenolate ion nucleophiles: the brønsted β for the calibrating equilibrium for N-methylisonicotinyl (4-carbonyl-N-methylpyridinium) transfer

Rate constants have been measured for the reaction of substituted phenolate ions with aryl acetate esters and with aryl N-methylisonicotinate esters in aqueous solution. A new method is demonstrated for determining βeq for group transfer from 4-nitrophenyl esters; it employs the rate constant for the reaction of 2,6-difluorophenolate ion with substituted phenyl ester as a surrogate for the reactivity of the 4-nitrophenolate ion and yields βeq= 1.55 for the N-methylisonicotinyl transfer reaction. The Bronsted-type plot of the rate constant for phenolate ion attack on 4-nitrophenyl N-methylisonicotinate is linear over a range of pKa values from 5.5 to 10 and provides good evidence for a concerted displacement mechanism for this reaction. The reactivity of the N-methylisonicotinate esters to phenolate ions is some 300 times larger than that of the corresponding acetate esters but the larger βnuc value (0.90 compared with 0.74) suggests a ‘later’ transition structure. Calibration of the β values with the corresponding βeq gives a Leffler αnuc= 0.58 and 0.42 for N-methylisonicotinate and acetate respectively, which contrasts with the order expected from reactivity–selectivity. The tighter transition structure indicated by comparison of these α values is explained by a less favourable acylium ion in the case of the N-methylisonicotinyl transfer reaction.

Aminolysis of sulfamate esters in non-aqueous solvents. Evidence consistent with a concerted E2-type mechanism

Aminolysis of 4-nitrophenyl sulfamate, N-phenylmethylsulfamate and N-phenylsulfamate esters in chloroform and acetonitrile at 37 °C using a series of pyridine, quinuclidine and cyclic amine bases has been studied. Second-order kinetics were observed and k2 constants were obtained from plots of kobsvs. [amine]. Bronsted-type plots gave slopes of ∼ 0.27 for the pyridines and ∼ 0.5 for the stronger bases. The failure of the N,N-dimethylsulfamate, Me2NSO2OC6H4NO2-4, to react and the absence of steric effects when hindered pyridines were used support an elimination rather than a substitution mechanism and thus the amines act as general base catalysts. Negative entropies of activation support a bimolecular mechanism and an isokinetic temperature of 541 K was obtained from a plot of ΔH‡vs.ΔS‡. A β1g of –1.25 (ρ=+2.8) has been obtained for reaction of a series of five N-phenylmethyl esters, PhCH2NHSO2OC6H4X (X = 4-NO2, 3-NO2, 3-Cl, 4-Cl and H with tert-butylamine in chloroform and a ρacyl, of –0.91 for reaction of the phenyl esters, XC6H4NHSO2OC6H4X (X = 4-MeO, 4-Me, 3-Me, H and 3-Cl) with 4-dimethylaminopyridine in chloroform. These reactions are seen as being E2 type processes with some ElcB-like character but they are not thought to involve N-sulfonylamines, RNSO2.

A calixresorcinarene provides the framework for an artificial esterase

An octa(dimethylaminopropyl)calixresorcin[4]arene (1c) is a primitive artificial esterase for 4-nitrophenyl esters.

Substrate dependence of amiloride- and soman-induced conformation changes of butyrylcholinesterase as evidenced by high-pressure perturbation

Previous results on butyrylcholinesterase-catalyzed hydrolysis of o-nitrophenylbutyrate in the presence of soman, an irreversible inhibitor of cholinesterases, suggested that reversible binding of soman preceding enzyme phophonylation induced a new enzyme conformational state (E′). The purpose of the present study was to determine whether this effect depends on soman itself or is dependent on the presence and nature of substrate or ligand. First, we examined the effect of amiloride, a reversible cholinesterase effector, upon the butyrylcholinesterase-catalyzed hydrolysis of nitrophenyl esters. The effect of amiloride was found to be dependent on the position ortho or para of the substrate nitro group: amiloride acts as a non-linear reversible activator of p-nitrophenyl ester hydrolysis and as a non-linear reversible inhibitor of o-nitrophenyl ester hydrolysis. Second, the effect of amiloride upon hydrolysis of o/ p-nitrophenylbutyrate was also studied under perturbing conditions, i.e., as a function of pressure (1–1600 bar) in the presence and absence of soman. Results show that the effect of reversible soman binding on butyrylcholinesterase activity in the presence of amiloride depends on the position of the substrate nitro group and amiloride concentration. Molecular modelling suggests that the presence of amiloride determines the orientation of ortho- and para-nitrophenyl esters in the active-site gorge. The nitro group of o-nitrophenylbutyrate interacts with the oxyanion hole via hydrogen bonds and its phenyl ring interacts with amiloride whose heterocycle faces Trp-82. The nitro group of p-nitrophenylbutyrate does not interact with the oxyanion hole but points towards Tyr-332; the phenyl ring of p-nitrophenylbutyrate interacts with amiloride but there is no steric constraint on the acyl chain. Thus, the network of interactions in ternary complexes is tighter with o-nitrophenylbutyrate as the substrate. There is no evidence for the existence of amiloride and/or soman-induced E′ state when p-nitrophenylbutyrate is the substrate. On the other hand, reversible binding of amiloride and/or soman induces new active conformational states that may be either binary (or ternary) enzyme-ligand complex or new free enzyme conformation resulting from long-lived ligand-induced enzyme conformational change when o-nitrophenylbutyrate is the substrate. These ligand-induced states are stabilized by high pressure.

Microgels with trialkylammonio side chains function as catalysts for the alkaline hydrolysis of substituted phenyl laurate esters

Microgels incorporating 2-(tetradecyldimethylammonio)ethyl methacrylate bromide have been prepared by emulsion polymerisation with methyl methacrylate, 2-ethoxyethyl methacrylate and ethylene glycol dimethacrylate in the molar ratio 0.15:0.45:0.30:0.10 respectively. The microgel solubilises aryl laurate esters in aqueous solution and catalyses the alkaline hydrolysis of these esters; the variation of rate constant with microgel concentration indicates a complexation phenomenon with the 4-nitrophenyl ester. The reactivity of the microgel sol with the laurate esters is similar to that of the analogous micellar system from cetyltrimethylammonium bromide (CTAB) described by Al-Awadi and Williams ( J. Org. Chem., 55 (1990) 2001). The rate constant for the microgel-catalysed reaction of hydroxide ion with the substituted phenyl laurate esters obeys the Brønsted correlation: logk OH cat = −0.53 pK a+4.49 The value of the Brønsted β 1g (−0.53) is similar to that observed for CTAB-catalysis and for uncatalysed alkaline hydrolysis; it indicates that the catalytic site of ester hydrolysis resides in an aqueous-like region of the microgel.

Purification and properties of three esterases from Brevibacterium sp. R312

C. LAMBRECHTS, J. ESCUDERO AND P. GALZY. 1995. The esterases of Brevibacterium sp. R312 were found to have an intracellular location. Electrophoresis of lysed cell supernatant fluids revealed seven bands of esterase activity in the presence of α‐naphthyl acetate. Eight esterases were separated by anion exchange chromatography. The three main esterases (esterase 4b, 2 and 4a) of Brevibacterium sp. R312 were purified. The molar masses, the pH optima, the temperature optima and heat stabilities were determined. Esterase 2 differed from the two others in sensitivity to inhibitors. Esterase 4b differed from esterases 2 and 4a in its substrate specificity. This enzyme hydrolyses aliphatic and nitrophenyl esters. The spectrum of activity of the two other esterases is narrower. They hydrolysed only naphthyl esters and, in the case of esterase 2, tributyrate and ethyl butyrate.

The enzymatic hydrolysis of a synthetic biomembrane: a new substrate for cholesterol and carboxyl esterases.

With the introduction of artificial implant devices, a new host of biomembrane-like structures have been introduced into the bio-media made up of the synthetic matrix, adsorbed proteins and lipids. Lysosomal hydrolases, e.g. cholesterol esterase (CE), are implicated during the tissue response near the tissue-implant interface. The enzymatic attack on a radiolabelled 'hybrid biomembrane', a polyester-urethane (PUU-CAP), was investigated using two esterases. Membrane stability was monitored by release of radiolabelled molecules. Although some radioactivity was released by buffer controls, upon the addition of CE, a burst of radiolabel release occurred which was due to an enzymatic reaction that could be saturated and inhibited by the specific esterase inhibitor, phenylmethylsulfonylfluoride. Carboxyl esterase (CXE) incubation with PUU-CAP caused less radiolabel release than CE which was similar to the latter's activity when common nitrophenyl ester substrates were used. When a factorial analysis was performed, it was found that side chain length for the common substrates was twice more important for CE, than CXE activity. This would suggest that CE activity is greater for substrates which have spacer segments between potential ester-carbonyl cleavage sites and the rigid ring structure.

New Insights on the Specificity of Penicillin Acylase

In contrast with the general thought that penicillin G acylases (PGAs) were only able to hydrolyse amides or esters of higly hydrophobic acids, we have demonstrated that the PGA from Kluyvera citrophila catalysed the hydrolysis of 4-nitrophenyl esters of acetic, propionic, butyric and valeric acids. Values of kcat. and kcat./Km were greatest for the first compound and less than values for benzylpenicillin by factors of 30 and 7, respectively. 4-Nitrophenyl acetate was hydrolysed faster than 2-nitrophenyl acetate but slower than phenyl acetate. The pH dependence of the reaction was similar to that of benzylpenicillin. Several experiments showed that hydrolysis of 4-nitrophenyl acetate was not catalysed by contaminating esterase activity. The implications for the structural basis of substrate binding are discussed. These substrates open, for the first time, a way to investigate the kinetic parameters of PGA at the presteady-state and provides a new perspective about the role of PGA in nature.