Formation Of Isoindole Derivative Research Articles

Purification and properties of a low molecular weight 1,4-β- d-glucan glucohydrolase having one active site for carboxymethyl cellulose and xylan from an alkalothermophilic Thermomonospora sp.

A low molecular weight 1,4-β- d-glucan glucohydrolase from an extracellular culture filtrate of Thermomonospora sp. was purified to homogeneity. The molecular weight of the purified enzyme was 14.2 kDa by MALDI-TOF analysis and is in agreement with SDS–PAGE and gel filtration chromatography. The purified enzyme exhibited both endocarboxymethyl cellulase and endoxylanase activities. A kinetic method was employed to study the active site of the enzyme that hydrolyzes both carboxymethyl cellulose and xylan. The experimental data coincide well with the theoretical values calculated for the case of a single active site. Conformation and microenvironment at the active site was probed with fluorescent chemo-affinity labeling using o-phthalaldehyde as the chemical initiator. Formation of isoindole derivative resulted in complete inactivation of the enzyme to hydrolyze both xylan and CMC as judged by fluorescence studies corroborating a single active site for the hydrolysis of xylan and CMC.

Studies on the inactivation of Leuconostoc mesenteroides NRRL B-512F dextransucrase by o-phthalaldehyde: evidence for the presence of an essential lysine residue at the active site.

The kinetics of inactivation of Leuconostoc mesenteroides NRRL B-512F dextransucrase by o-phthalaldehyde showed that the reaction followed pseudo-first order reaction. The loss of enzyme activity was concomitant with an increase in fluorescence at 417 nm indicating that the inhibition involved the reaction of an epsilon-amino and a thiol group of the enzyme leading to the formation of an isoindole derivative. The stoichiometry of inactivation showed that one isoindole derivative was formed per enzyme molecule. The substrates sucrose and glucose provided protection against o-phthalaldehyde inactivation which was also corroborated by fluorescence studies. Dextransucrase was not inactivated by 5,5'-dithiobis(2-nitrobenzoic acid), showing that the cysteine present in close proximity to the lysine is not essential for enzyme activity. Denaturation of dextransucrase by urea or heat treatment prior to o-phthalaldehyde addition resulted in a decrease of fluorescence intensity indicating that the native conformation of the enzyme is essential for isoindole derivative formation. These results established that a lysine residue is present at the active site and is essential for the activity of dextransucrase.

The cross-linking by o-phthalaldehyde of two amino acid residues at the active site of 6-phosphogluconate dehydrogenase.

o-phthalaldehyde inactivates homodimeric, NADP+ dependent, 6-phosphogluconate dehydrogenase from sheep liver, upon formation of a single isoindole derivative per enzyme subunit. This indicates that the thiol group of a cysteine residue or the epsilon-amino group of a lysine residue located within 3 A and crosslinked by the reagent is essential for catalysis. Fluorescence analyses of the modified enzyme suggest that the isoindole derivative forms at the binding site of the nicotinamide moiety of NADP+. The enzymes from Trypanosoma brucei and Lactococcus lactis are also inactivated suggesting a similar three-dimensional structure in this domain. The isoindole derivative does not form with two mutants of the T. brucei enzyme (Lys185His and Lys185Leu), this allowing to identify not only the lysine but also the cysteine involved in the cross-linking. The formation of the isoindole derivative inactivates not only the oxidative decarboxylation, but also two partial reactions catalysed by the enzyme.

Identification of cysteine and lysine residues present at the active site of beef liver glutamate dehydrogenase by o-phthalaldehyde

Beef liver glutamate dehydrogenase (GDH) is inactivated by the bifunctional reagent, o-phthalaldehyde. The initial rate of inactivation follows pseudo first-order kinetics. The reaction of the enzyme with o-phthalaldehyde results in isoindole derivative formation which is characterized by typical fluorescence emission and excitation maximum at 410 nm and 337 nm, respectively. The inactivation of GDH by o-phthalaldehyde is partially prevented by α-ketoglutaric acid, whereas NADH does not provide any protection. This clearly indicates that cysteine and lysine residues are located near the α-ketoglutaric acid binding center. The dissociation constant of 2.2 mM was obtained for enzyme-α-ketoglutaric acid complex. Stoichiometry of o-phthalaldehyde binding with glutamate dehydrogenase showed that the formation of approximately one isoindole derivative per subunit of glutamate dehydrogenase is accompanied by complete loss of activity.

S1 nuclease: immunoaffinity purification and evidence for the proximity of cysteine 25 to the substrate binding site.

A simple procedure, involving heat treatment, gel filtration on Sephadex-G 100 followed by chromatography on anti-S1 nuclease antibodies bound to Sepharose, was developed for purification of S1 nuclease to homogeneity with an overall yield of 72%. S1 nuclease was rapidly inactivated, at pH 6.0 and 37 degrees C, in presence of o-phthalaldehyde. Kinetic analysis of o-phthalaldehyde medicated inactivation showed that the reaction followed pseudo-first-order kinetics and the loss of enzyme activity was due to the formation of a single isoindole derivative per molecule of the enzyme. Absorbance and fluorescence spectrophotometric data also gave similar results. The isoindole derivative formation, as a result of o-phthalaldehyde treatment is known to occur through crosslinking of the thiol group of cysteine and the epsilon-amino group of lysine, situated in close proximity in the native enzyme. Since, modification of the only available cysteine residue (Cys25) did not affect the catalytic activity of the enzyme, the o-phthalaldehyde mediated inactivation of S1 nuclease is due to the modification of lysine. Substrates of S1 nuclease, namely ssDNA, RNA, 3'AMP, could protect the enzyme against o-phthalaldehyde mediated inactivation. Moreover, the modified enzyme (having very little catalytic activity) showed a significant decrease in its ability to bind 5'AMP, a competitive inhibitor of S1 nuclease, suggesting that the modification has occurred at the substrate binding site. The above results point towards the presence of cysteine 25 in close proximity to the substrate binding site.

Reactivity of essential cysteine and lysine residues present at the catalytic domain of pig heart mitochondrial malate dehydrogenase.

Pig heart mitochondrial malate dehydrogenase was inactivated very rapidly by omicron-phthalaldehyde as compared to 5,5'-dithio bis(2-nitrobenzoic acid) and pyridoxal 5'-phosphate. The omicron-phthalaldehyde reaction followed pseudo first order kinetics, and a second order rate constant of 38 M-1 S-1 was obtained. Cysteine and lysine residues participating in the omicron-phthalaldehyde reaction are located in the NADH binding region of malate dehydrogenase as shown by protection experiments. The decrease in the rate of inactivation in the presence of NADH was used to determine the dissociation constant of the enzyme-NADH complex. pH dependent studies and molar transition energy calculations of the omicron-phthalaldehyde-inactivated enzyme have indicated that cysteine and lysine residues involved in the isoindole derivative formation are located in a hydrophobic environment at the coenzyme binding site.

Kinetic fluorimetric determination of aminoglycoside antibiotics by use of OPA and N-acetylcysteine as reagents

The use of N-acetylcysteine for the kinetic fluorimetric determination of aminoglycoside antibiotics by reaction with o-phthaldialdehyde (OPA) in a basic medium was studied. The results obtained show that this thiol is a satisfactory substitute for 2-mercaptoethanol as reagent for the formation of isoindole derivatives of compounds containing primary amines such as amikacin, kanamicin and tobramycin, while avoiding its unpleasant odour. Due to the fast formation of the fluorescent derivatives the stopped-flow mixing technique must be used in order to acquire analytical kinetic data. This allowed the development of a kinetic automatic method for the determination of amikacin applicable to routine analyses. The initial rate was measured within ca. 5 s and the calibration graph was linear over the range 0.05–30.0 μg mL−1. The method was satisfactorily applied to the determination of amikacin in two pharmaceutical preparations with a mean recovery of 98.7%.

Investigation of the nature of o-phthalaldehyde reaction with octopine dehydrogenase.

The effect of o-phthalaldehyde on octopine dehydrogenase inactivation has been studied. o-Phthalaldehyde binds to the proximal cysteine and lysine residues of the enzyme leading to the formation of isoindole derivative. Double inhibition studies with o-phthalaldehyde and p-chloromercuricphenyl sulfonic acid have indicated that o-phthalaldehyde does not bind to the functional cysteine present at the active site. Protection experiments have shown that L-arginine prevented o-phthalaldehyde inactivation. This could be only due to the reaction of the amino group of L-arginine with o-phthalaldehyde as per the mechanism proposed elsewhere since L-arginine cannot bind to the enzyme prior to NADH. Other substrates such as pyruvate oR NADH could not prevent the o-phthalaldehyde reaction with the enzyme. Fluorescence spectral studies demonstrated that in the presence of externally added amino acid no isoindole derivative formation occurs. However, a characteristic isoindole derivative is formed in the presence of beta-mercaptoethanol although the enzyme does not lose its activity. This indicated that o-phthalaldehyde can bind with lysine of the enzyme and thiol of externally added beta-mercaptoethanol. Pyridoxal 5'-phosphate, a lysine specific reagent also binds to the enzyme giving the characteristic absorption and fluroescence peak at 325 nm and 395 nm respectively. However, no loss of enzyme activity was observed. On the basis of these experiments we would suggest that o-phthalaldehyde binds to non-essential cysteine and lysine residues present in close proximity which results in conformational changes leading to enzyme inactivation.

Evidence for proximal cysteine and lysine residues present at the nucleotide domain of rabbit muscle creatine kinase

Inactivation of rabbit muscle creatine kinase by o-phthalaldehyde was investigated. The loss of enzyme activity was concomitant with the increase in fluorescence intensity at 410 nm. The modified enzyme showed a characteristic absorption peak at 336 nm. These evidences suggested that the mechanism of inhibition of creatine kinase by o-phthalaldehyde involves binding of the thiol and the ϵ-amino group of enzyme leading to the formation of isoindole derivative. None of the substrate, except Mg-ATP, provided protection against o-phthalaldehyde inhibition. This was corroborated by fluorescence studies. Double inhibition experiments showed that p-chloromercuricphenyl sulphonic acid, a thiol-specific reagent, binds to the same cysteine which is also involved in the o-phthalaldehyde reaction. Stoichiometric results indicated that 2 mol of o-phthalaldehyde were incorporated per mole of enzyme molecule upon complete inactivation. Denaturation of creatine kinase by urea or heat treatment prior to o-phthalaldehyde addition resulted in the decrease of fluorescence intensity indicating that native conformation of the enzyme is essential for isoindole derivative formation.

Inactivation of chicken mitochondrial phosphoenolpyruvate carboxykinase by o-phthalaldehyde.

Chicken liver mitochondrial phosphoenolpyruvate carboxykinase is inactivated by o-phthalaldehyde. The inactivation followed pseudo first-order kinetics, and the second-order rate constant for the inactivation process was 29 M-1 s-1 at pH 7.5 and 25 degrees C. The modified enzyme showed maximal fluorescence at 427 nm upon excitation at 337 nm, consistent with the formation of isoindole derivatives by the cross-linking of proximal cysteine and lysine residues. Activities in the physiologic reaction and in the oxaloacetate decarboxylase reaction were lost in parallel upon modification with o-phthalaldehyde. Plots of (percent of residual activity) versus (mol of isoindole incorporated/mol of enzyme) were biphasic, with the initial loss of enzymatic activity corresponding to the incorporation of one isoindole derivative/enzyme molecule. Complete inactivation of the enzyme was accompanied by the incorporation of 3 mol of isoindole/mol of enzyme. beta-Sulfopyruvate, an isoelectronic analogue of oxaloacetate, completely protected the enzyme from reacting with o-phthalaldehyde. Other substrates provided protection from inactivation, in decreasing order of protection: oxaloacetate greater than phosphoenolpyruvate greater than MgGDP, MgGTP greater than oxalate. Cysteine 31 and lysine 39 have been identified as the rapidly reacting pair in isoindole formation and enzyme inactivation. Lysine 56 and cysteine 60 are also involved in isoindole formation in the completely inactivated enzyme. These reactive cysteine residues do not correspond to the reactive cysteine residue identified in previous iodoacetate labeling studies with the chicken mitochondrial enzyme (Makinen, A. L., and Nowak, T. (1989) J. Biol. Chem. 264, 12148-12157). Protection experiments suggest that the sites of o-phthalaldehyde modification become inaccessible when the oxaloacetate/phosphoenolpyruvate binding site is saturated, and sequence analyses indicate that cysteine 31 is located in the putative phosphoenolpyruvate binding site.

Formation of Isoindole Derivatives of Sulfidopeptide Leukotrienes by Reaction with O-Phthalaldehyde and Separation by Reverse Phase High Performance Liquid Chromatography

Abstract A method is described for the formation of fluorescent conjugates of the sulfidopeptide leukotrienes (LTC4, LTD4, and LTE4) by reaction of the primary amine moiety of these metabolites with o-phthalaldehyde. Separation of the fluorescent derivatives was achieved by reverse-phase high performance liquid chromatography in less than 30 minutes using a convex gradient of methanol-50 mM Na Acetate-5% Tetrahydrofuran pH 5.5. Detection limits realized under the conditions described were 0.35 ng, 3.8 ng and 3.7 ng for LTC4, LTD4, and LTE4, respectively. This represents an Increased sensitivity over detection of these metabolites by ultra-violet spectroscopy. The leukotriene-OPA derivatives are fully stable for 50 minutes at 23°C and for at least 4 hours at 0°C. The method is applied to the detection of LTC4 generated by zymosan stimulated murine peritoneal macrophages.

Formation of isoindole derivatives by reaction of phthalonitrile with sodium methoxide

It is shown by means of IR spectra that the reaction of phthalonitrile with sodium methoxide in benzene in the presence of traces of water or methanol proceeds via a series-parallel mechanism in two steps to give cyclic products. The proposed structure of the intermediate is an oligomeric isbindolenine chain with terminal NNa and OCH3 groups. The final product is the sodium salt of monomethoxyiminoisoindolenine. The analogous reaction in methanol proceeds at a higher rate that makes it impossible to isolate or detect the intermediate; the final product is 1,1-dimethoxy-3-iminoisoindoline.