Inhibitors of microsomal oxidations in insect homogenates

1. Homogenates of insect tissues were assayed for enzymes capable of oxidizing p-nitrotoluene to p-nitrobenzoic acid. 2. Locust fat-body homogenate 10000g supernatant was an effective enzyme and required no added cofactors. 3. Homogenates of other insects or locust organs and 10000g sediment from locust fat-body were not active and inhibited microsomal oxidations carried out by locust fat-body or rabbit liver enzyme. 4. Inhibitory power was high in homogenates of whole flies and of fly heads or thoraces. 5. Inhibition appeared to involve both irreversible inactivation of enzyme and the removal of essential cofactors.     

Delta(2)-oxazolines-1,3 and N-acylaziridines as potential proinsecticides of carboxylic acids: V. Direct thin-layer chromatography monitoring of the metabolism in locust tissues

Modern thin-layer chromatography (TLC) was used for the evaluation of Δ²-oxazolines-1,3 I and N-acylaziridine VII structures, as potential proinsecticides of carboxylic acids III. Thus the unmasking² of the active principles III from Δ²-oxazolines-1,3 Ia–c and N-acylaziridine VIIc was monitored by spotting aliquots directly onto RP-18 TLC plates, without any sample pretreatment during in vitro assays performed in concentrated locust tissues. To achieve a good separation of carboxylate IIIa from endogenous components of the tissues, a short preliminary development with methanol or ion-pairing was necessary. From UV–TLC chromatograms (densitograms) it appeared that in a phosphate buffer at pH 7.4, the oxazoline Ia with a C₂ substituent devoid of α-ramification or α,β-insaturation hydrolysed slowly into the corresponding β-hydroxylamide VIa and intermediate aminoester Va. Significantly, locust mesenteron (or fat body) efficiently triggered the unmasking of IIIa, a transformation which corresponds to the expected proinsecticide behavior of Ia. Conducting TLC monitoring in the same locust tissues also revealed that the oxazolines Ib and Ic with an α-ramification and an α,β-insaturation, respectively, cannot be considered as proinsecticides of the corresponding carboxylic acids IIIb and IIIc. In contrast, the N-acylaziridine VIIc appeared as a convenient proinsecticide structure for masking the carboxylic acid IIIc.     

Trypsin-specific acyl-4-guanidinophenyl esters for alpha-chymotrypsin-catalysed reactions computational predictions, hydrolyses, and peptide bond formation.

The function of acyl-4-guanidinophenyl esters as substrate mimetics for the serine protease alpha-chymotrypsin was investigated by protein-ligand docking, hydrolysis, and acyl transfer experiments. On the basis of protein-ligand docking studies, the binding and hydrolysis properties of these artificial substrates were estimated. The predictions of the rational approach were confirmed by steady-state hydrolysis studies on 4-guanidinophenyl esters derived from coded amino acids (which alpha-chymotrypsin is not specific for), noncoded amino acids, and even simple carboxylic acid moieties. Enzymatic peptide syntheses qualify these esters as suitable acyl donors for the coupling of acyl components far from the natural enzyme specificity, thus considerably expanding the synthetic utility of alpha-chymotrypsin.     

A New Enzymatic Reaction: Enzyme Catalyzed Ammonolysis of Carboxylic Esters

A new enzymatic reaction of carboxylic esters and ammonia (ammonolysis) was studied. This reaction provides a synthetically useful and mild alternative for the synthesis of amides. Several lipases and one esterase acted as catalyst. Ammonolysis of esters of chiral carboxylic acids gave higher ee values than hydrolysis under comparable reaction conditions. Furthermore, consecutive enzymatic esterification and ammonolysis provided a convenient one-pot synthesis of carboxylic amides from carboxylic acids.     

Interaction of alpha-chymotrypsin with dimethylsulfoxide: a change of substrate could change the mechanism of interaction

The kinetic behavior of alpha-chymotrypsin was studied in water-DMSO mixtures at concentrations of the organic solvent that do not cause irreversible denaturation of the enzyme. Various substrates (N-substituted derivatives of L-tyrosine) were found to display substantially different kinetic patterns of interaction with alpha-chymotrypsin, which can be described by totally different kinetic schemes. The differences were ascribed to competition between the N-acyl group of the substrate and the DMSO molecule at the S2-site of substrate binding to the active site of the enzyme.     

Synthesis of epimers of L-cyclopentenylglycine using enzymatic resolution

Both epimers of the naturally occurring nonproteinogenic amino acid L-cyclopentenylglycine, (2S,1'S)- and (2S, 1'R)-2-(cyclopent-2'-enyl)glycine, were obtained via a procedure involving condensation of 3-chlorocyclopentene with diethyl acetylaminomalonate, deethoxycarbonylation, chromatographic separation of the resulting two pairs of enantiomers, and enzymatic resolution of the racemates employing enantioselective hydrolysis of the ethyl ester group with alpha-chymotrypsin. The method was used for preparation of (13)C-labeled compounds of interest for biosynthetic tracer experiments. Enantiomeric purity of the products was determined by chiral HPLC on a Crownpak CR(+) column. The biologically active (2S,1'R) isomer was obtained as a pure compound and characterized for the first time. The (2R,1'R) and (2R,1'S) isomers were obtained as N-acetyl ethyl ester derivatives.     

Lipase-mediated Resolution of Branched Chain Fatty Acids

Branched chain fatty acids (BCFAs) are fatty acids substituted with alkyl groups. Many of them are chiral and therefore occur in two enantiomeric forms. This review describes their occurrence in Nature, their biosynthesis, their properties as flavours, and their enzymatic kinetic resolution. Many lipases are able to separate the enantiomers of BCFAs, in hydrolysis, esterification or transesterification reactions. Very often, the stereoselectivity of these reactions is remarkably high, even when the chiral carbon atom is remote from the carboxylic acid group.     

Quantitative analysis of enantioselective enzymatic hydrolysis with non-enantioselective removal of chiral products

Kinetic resolution of racemic compounds by enzymatic hydrolysis with non-enantioselective separation of enantiomer products via a separator or ion-pair formation has been quantitatively analyzed. Theoretical results indicate that the removal of chiral products has profound effects on improving the conversion and enantiomeric excess for the desired chiral substrate or product. The analysis was confirmed from lipase-catalyzed hydrolysis of racemic methyl 2-chloropropionate in the presence of pyrrolidine in buffer saturated dichloromethane.     

Enhancing the enzymatic hydrolysis of lignocellulosic biomass by increasing the carboxylic acid content of the associated lignin

To assess the effects that the physical and chemical properties of lignin might have on the enzymatic hydrolysis of pretreated lignocellulosic substrates, protease treated lignin (PTL) and cellulolytic enzyme lignin (CEL) fractions, isolated from steam and organosolv pretreated corn stover, poplar, and lodgepole pine, were prepared and characterized. The adsorption of cellulases to the isolated lignin preparations corresponded to a Langmuir adsorption isotherm. It was apparent that, rather than the physical properties of the isolated lignin, the carboxylic acid functionality of the isolated lignin, as determined by FTIR and NMR spectroscopy, had much more of an influence when lignin was added to typical hydrolysis of pure cellulose (Avicel). An increase in the carboxylic content of the lignin preparation resulted in an increased hydrolysis yield. These results suggested that the carboxylic acids within the lignin partially alleviate non-productive binding of cellulases to lignin. To try to confirm this possible mechanism, dehydrogenative polymers (DHP) of monolignols were synthesized from coniferyl alcohol (CA) and ferulic acid (FA), and these model compounds were added to a typical enzymatic hydrolysis of Avicel. The DHP from FA, which was enriched in carboxylic acid groups compared with the DHP from CA, adsorbed a lower mount of cellulases and did not decrease hydrolysis yields when compared to the DHP from CA, which decreased the hydrolysis of Avicel by 8.4%. Thus, increasing the carboxylic acid content of the lignin seemed to significantly decrease the non-productive binding of cellulases and consequently increased the enzymatic hydrolysis of the cellulose.     

Purification and characterization of thermostable beta-mannanase and alpha-galactosidase from Bacillus stearothermophilus.

Bacillus stearothermophilus secretes beta-mannanase and alpha-galactosidase enzymatic activities capable of hydrolyzing galactomannan substrates. Expression of the hemicellulase activities in the presence of locust bean gum was sequential, with mannanase activity preceding expression of alpha-galactosidase activity. The hemicellulase activities were purified to homogeneity by a combination of ammonium sulfate fractionation, gel filtration, hydrophobic interaction chromatography, and ion-exchange and chromatofocusing techniques. The purified beta-D-mannanase is a dimeric enzyme (162 kilodaltons) composed of subunits having identical molecular weight (73,000). Maximal activity did not vary between pH 5.5 and 7.5. The beta-D-mannanase activity exhibited thermostability, retaining nearly full activity after incubation for 24 h at 70 degrees C and pH 6.5. The enzyme displayed high specificity for galactomannan substrates, with no-secondary xylanase or cellulase activity detected. Hydrolysis of locust bean gum yielded short oligosaccharides compatible with an endo mode of substrate depolymerization. Initial rate velocities of the mannanase activity displayed substrate inhibition and yielded estimates for Vmax and Km of 455 +/- 60 U/mg and 1.5 +/- 0.3 mg/ml, respectively, at 70 degrees C and pH 6.5. The alpha-galactosidase activity corresponded to a trimeric enzyme (247 kilodaltons) having subunits of identical molecular weight (82,000). The alpha-galactosidase had maximal activity at pH 7 to 7.5 and retained full activity after 24 h of incubation at 60 degrees C. The enzyme had only limited activity on galactomannan substrates as compared with hydrolysis of p-nitrophenyl alpha-D-galactose. Kinetics of p-nitrophenyl alpha-D-galactose hydrolysis yielded linear reciprocal plots corresponding to Vmax and Km of 195 +/- 10 U/mg and 0.25 +/- 0.02 mM, respectively, at 60 degrees C and pH 7. The characterization of the mannanase activity is consistent with its potential use in enzymatic bleaching of softwood pulps.     

Lipase-mediated Resolution of Branched Chain Fatty Acids

Branched chain fatty acids (BCFAs) are fatty acids substituted with alkyl groups. Many of them are chiral and therefore occur in two enantiomeric forms. This review describes their occurrence in Nature, their biosynthesis, their properties as flavours, and their enzymatic kinetic resolution. Many lipases are able to separate the enantiomers of BCFAs, in hydrolysis, esterification or transesterification reactions. Very often, the stereoselectivity of these reactions is remarkably high, even when the chiral carbon atom is remote from the carboxylic acid group.     

Triazene drug metabolites. Part 17: Synthesis and plasma hydrolysis of acyloxymethyl carbamate derivatives of antitumour triazenes

A series of 3-acyloxymethyloxycarbonyl-1-aryl-3-methyltriazenes 5 was synthesised by the sequential reaction of 1-aryl-3-methyltriazenes with (i) chloromethyl chloroformate, (ii) NaI in dry acetone, and (iii) either the silver carboxylate or the carboxylic acids in the presence of silver carbonate. The hydrolysis of these compounds was studied in pH 7.7 isotonic phosphate buffer and in human plasma. Triazene acyloxycarbamates demonstrated their ability to act as substrates for plasma enzymes. For compound 5f, a pH-rate profile was obtained which showed the hydrolysis to involve acid-base catalysis. The reaction is also buffer catalysed. Thus, at pH 7.7, pH-independent, base-catalysed and buffer-catalysed processes all contribute to the hydrolysis reaction. The sensitivity of the hydrolysis reaction to various structural parameters in the substrates indicates that hydrolysis occurs at the ester rather than the carbamate functionality. In plasma, the rates of hydrolysis correlate with partition coefficients, the most lipophilic compounds being the most stable. An aspirin derivative suffers two consecutive enzymatic reactions, the scission of the aspirin acetyl group being followed by the scission of the acyloxy ester group. These results indicate that triazene acyloxymethyl carbamates are prodrugs of the antitumour monomethyltriazenes. They combine chemical stability with a rapid enzymatic hydrolysis, and are consequently good candidates for further prodrug development. Moreover, this type of derivative allowed the synthesis of mutual prodrugs, associating the antitumour monomethyltriazenes with anti-inflammatory NSAIDs as well as with the anticancer agent butyric acid.     

Purification and characterization of thermostable beta-mannanase and alpha-galactosidase from Bacillus stearothermophilus

Bacillus stearothermophilus secretes beta-mannanase and alpha-galactosidase enzymatic activities capable of hydrolyzing galactomannan substrates. Expression of the hemicellulase activities in the presence of locust bean gum was sequential, with mannanase activity preceding expression of alpha-galactosidase activity. The hemicellulase activities were purified to homogeneity by a combination of ammonium sulfate fractionation, gel filtration, hydrophobic interaction chromatography, and ion-exchange and chromatofocusing techniques. The purified beta-D-mannanase is a dimeric enzyme (162 kilodaltons) composed of subunits having identical molecular weight (73,000). Maximal activity did not vary between pH 5.5 and 7.5. The beta-D-mannanase activity exhibited thermostability, retaining nearly full activity after incubation for 24 h at 70 degrees C and pH 6.5. The enzyme displayed high specificity for galactomannan substrates, with no-secondary xylanase or cellulase activity detected. Hydrolysis of locust bean gum yielded short oligosaccharides compatible with an endo mode of substrate depolymerization. Initial rate velocities of the mannanase activity displayed substrate inhibition and yielded estimates for Vmax and Km of 455 +/- 60 U/mg and 1.5 +/- 0.3 mg/ml, respectively, at 70 degrees C and pH 6.5. The alpha-galactosidase activity corresponded to a trimeric enzyme (247 kilodaltons) having subunits of identical molecular weight (82,000). The alpha-galactosidase had maximal activity at pH 7 to 7.5 and retained full activity after 24 h of incubation at 60 degrees C. The enzyme had only limited activity on galactomannan substrates as compared with hydrolysis of p-nitrophenyl alpha-D-galactose. Kinetics of p-nitrophenyl alpha-D-galactose hydrolysis yielded linear reciprocal plots corresponding to Vmax and Km of 195 +/- 10 U/mg and 0.25 +/- 0.02 mM, respectively, at 60 degrees C and pH 7. The characterization of the mannanase activity is consistent with its potential use in enzymatic bleaching of softwood pulps.     

A comparison of the properties of the pyruvate kinases of the fat body and flight muscle of the adult male desert locust

1. The pyruvate kinases of the desert locust fat body and flight muscle were partially purified by ammonium sulphate fractionation. 2. The fat-body enzyme is allosterically activated by very low (1mum) concentrations of fructose 1,6-diphosphate, whereas the flight-muscle enzyme is unaffected by this metabolite at physiological pH. 3. Flight-muscle pyruvate kinase is activated by preincubation at 25 degrees for 5min., whereas the fat-body enzyme is unaffected by such treatment. 4. Both enzymes require 1-2mm-ADP for maximal activity and are inhibited at higher concentrations. With the fat-body enzyme inhibition by ADP is prevented by the presence of fructose 1,6-diphosphate. 5. Both enzymes are inhibited by ATP, half-maximal inhibition occurring at about 5mm-ATP. With the fat-body enzyme ATP inhibition can be reversed by fructose 1,6-diphosphate. 6. The fat-body enzyme exhibits maximal activity at about pH7.2 and the activity decreases rapidly above this pH. This inactivation at high pH is not observed in the presence of fructose 1,6-diphosphate, i.e. maximum stimulating effects of fructose 1,6-diphosphate are observed at high pH. The flight-muscle enzyme exhibits two optima, one at about pH7.2 as with the fat-body enzyme and the other at about pH8.5. Stimulation of the enzyme activity by fructose 1,6-diphosphate was observed at pH8.5 and above.     

Substrate effects on the enzymatic activity of alpha-chymotrypsin in reverse micelles

Six different substrates have been used for measuring the activity of alpha-chymotrypsin in reverse micelles formed by sodium bis(2-ethylhexyl) sulfosuccinate (AOT) in isooctane. The substrates were glutaryl-Phe p-nitroanilide, succinyl-Phe p-nitroanilide, acetyl-Phe p-nitroanilide, succinyl-Ala-Ala-Phe p-nitroanilide, succinyl-Ala-Ala-Pro-Phe p-nitroanilide and acetyl-Trp methyl ester. It has been shown that the dependence of the kinetic constants (kcat and Km) on the water content of the system, on wo (= [H2O]/[AOT]), is different for the different substrates. This indicates that activity-wo profiles for alpha-chymotrypsin in reverse micelles not only reflect an intrinsic feature of the enzyme alone. For the p-nitroanilides it was found that the lower kcat (and the higher Km) in aqueous solution, the higher kcat as well as Km in reverse micelles. "Superactivity" of alpha-chymotrypsin could only be found with the ester substrate and with relatively "poor" p-nitroanilides. The presence of a negative charge in the substrate molecule is not a prerequisite for alpha-chymotrypsin to show "superactivity".     

N-anthraniloylation converts peptide p-nitroanilides into fluorogenic substrates of proteases without loss of their chromogenic properties

By simple substitution of an N-acyl group for the anthraniloyl(o-aminobenzoyl) group, chromogenic p-nitroanilide substrates are converted into highly sensitive fluorogenic substrates of proteases. The fluorescence of the anthraniloyl group is completely quenched by the p-nitroanilide moiety in the intact substrates and is released during their enzymatic hydrolysis. The approach is exemplified by the synthesis of anthraniloyl-Phe p-nitroanilide, anthraniloyl-Lys p-nitroanilide, and anthraniloyl-Gly-Gly-Phe p-nitroanilide as substrates for chymotrypsin, trypsin, and alkaline mesentericopeptidase, respectively. The kinetic parameters of these substrates are slightly better than those of similar derivatives bearing other acyl groups, suggesting that the enhanced sensitivity is completely due to the method of measurement. Since the conversion does not affect the chromogenic properties of the substrates, the same compounds can be used as usual p-nitroanilide substrates as well.     

A novel Dps-type protein from insect gut bacteria catalyses hydrolysis and synthesis of N-acyl amino acids

A novel type of a microbial N-acyl amino acid hydrolase (AAH) from insect gut bacteria was purified, cloned and functionally characterized. The enzyme was obtained from Microbacterium arborescens SE14 isolated from the foregut of larvae of the generalist herbivore Spodoptera exigua. The substrates of AAH are N-acyl-glutamines previously reported to elicit plant defence reactions after introduction into the leaf during feeding. The isolated AAH catalyses the hydrolysis of the amide bond (K(m) = 36 micromol l(-1)) and, less efficient, the formation (K(m) = 3 mmol l(-1)) of the elicitor active N-acyl amino acids. The AAH from M. arborescens SE14 shows no homology to known fatty acyl amidases (EC 3.5.1.4) but belongs to the family of Dps proteins (DNA-binding protein from starved cell). In line with other DPS proteins AAH is a homododecamer (monomer 17 181 Da) and contains iron atoms (c. 1-16 iron atoms per subunit). Unlike genuine DPS proteins the enzyme does not significantly bind DNA. Amino acid hydrolase is the first member of the DPS family that catalyses the cleavage or formation of amide bonds. The participation of a microbial enzyme in the homeostasis of N-acyl-glutamines in the insect gut adds further complexity to the interaction between plants and their herbivores.     

Use of N-benzoyl-L-tyrosine thiobenzyl ester as a protease substrate. Hydrolysis by alpha-chymotrypsin and subtilisin BPN.

In the course of searching for specific chromogenic substrates which might be useful in screening for protease-deficient mutants of Bacillus subtilis, we have developed a method for the synthesis of N-benzoyl-L-tyrosine thiobenzyl ester (BzTyrSBzl) in good yield. Spontaneous base hydrolysis of this thiol ester is low, but several serine proteases hydrolyze it readily. Spectrophotometric measurement of the hydrolysis of the ester in the presence of 5,5'-dithiobis(2-nitrobenzoic acid) provides a continuous assay for chymotrypsin as sensitive as any assay reported in the literature. Serine proteases which hydrolyze this substrate may be detected in polyacrylamide disc gels by incubation in the presence of nitro blue tetrazolium. Apparent Km values of 0.02 and 7 mM and kcat values of 37 S-1 and 126 S-1 were observed for the hydrolysis of BzTyrSBzl by alpha-chymotrypsin and subtilisin BPN', respectively. Additionally, 5 mM indole was observed to behave as a strict competitive inhibitor of the alpha-chymotrypsin-catalyzed hydrolysis of BzTyrSBzl but was observed to increase the maximal rate of hydrolysis of p-nitrophenyl acetate by alpha-chymotrypsin by 30%, as previously described. These data, the published data of other workers, and results from studies with molecular models of trypsin and subtilisin BPN' are used as the basis for describing more fully a secondary hydrophobic binding pocket on alpha-chymotrypsin. The pocket is immediately adjacent to the active site serine and is tentatively suggested to be composed of 4 aliphatic side chain residues and 2 glycine residues.     

Rapid screening of enzymes for the enzymatic hydrolysis of chiral esters in drug discovery

Thirty-five enzymes were rapidly screened for their ability to selectively hydrolyze chiral esters to their corresponding carboxylic acids for the efficient generation of chiral intermediates in drug discovery. Optimization of the enzymatic reactions at various incubation times was performed using a robotic liquid handler. Enantiomeric pairs of chiral esters and carboxylic acids were then analyzed simultaneously by chiral GC/MS in a single analysis. This analytical approach is particularly useful for compounds that do not possess a conjugated chromophore or are volatile and difficult to analyze by chiral HPLC/UV or HPLC/MS. The resulting data was used to determine enantiomeric excesses and percent conversions to the desired enantiomer of the carboxylic acid for the selection of efficient enzymes for bioconversions in drug discovery in a pharmaceutical company.     

Crosslinking of imprinted proteases to maintain a tailor-made substrate selectivity in aqueous solutions

A covalent method to keep imprinted properties of proteins stable in aqueous as well as in organic environment is described. To stabilize the ligand induced acceptance for D-configured substrates by alpha-chymotrypsin or subtilisin Carlsberg, each protein was first vinylated by acylation with itaconic anhydride. Then, the tailoring of the derivatized proteins by precipitation in the presence of N-acetyl-D-tryptophan from an aqueous medium with 1-propanol, and the subsequent crosslinking of the enzyme preparations with ethylene glycol dimethacrylate in cyclohexane was carried out. The crosslinked imprinted proteins (CLIPs) obtained catalyzed the hydrolysis of N-acetyl-D-tryptophan ethyl ester in phosphate buffer and the corresponding back reaction in cyclohexane, respectively. The repeated use of CLIP-alpha-chymotrypsin in D-ester hydrolysis was demonstrated. Furthermore, this particular CLIP-alpha-chymotrypsin showed no loss in activity when it subsequently was used in the synthesis of N-acetyl-D-tryptophan ethyl ester in cyclohexane again. In the case of D-ester hydrolysis the reaction rate acceleration (k(enz)/k(nonenz)) was in the same order of magnitude of about 10(4)-10(5) mM(-1) for the two CLIP-proteases. The results suggest that enzymes tailored by imprinting technique do not lose their induced "new" property in the presence of water when they are prepared according to the described vinylation/crosslinking method (CLIP technique).