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PubMed1993

Hydrolysis of some organophosphorus dichlorophenyl esters by hen brain homogenates and rabbit serum compared with hydrolysis of paraoxon.

The hydrolysis of four organophosphorus dichlorophenyl esters and of paraoxon was studied in hen brain homogenates and in rabbit serum. All compounds were hydrolysed by both preparations, but the rates were different in the two preparations. EDTA inhibited the hydrolysis almost completely in rabbit serum, but had only a small effect on the hydrolysis in hen brain homogenates.

Animals↗
PubMed1991

Hydrolysis of phosphatidylcholine by phospholipase D is a common response to mitogens which stimulate inositol lipid hydrolysis in Swiss 3T3 fibroblasts.

The stimulated hydrolysis of inositol lipids and phosphatidylcholine (PtdCho) by bombesin, [Arg8]vasopressin ([Arg8]Vp) and prostaglandin F2 alpha (PGF2 alpha) was analysed in Swiss 3T3 cells pre-labelled to isotopic equilibrium with either [methyl-3H]choline, myo-[2-3H]inositol or [9,10 (n)-3H]palmitic acid. All three agonists activated the phospholipase D-catalysed hydrolysis of PtdCho as determined by the release of [3H]choline (Cho) and the formation of [3H]phosphatidylbutanol (PtdBut). The release of [3H]choline by each agonist exhibited similar sensitivity to prolonged pre-exposure to the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA). The release of [3H]choline exhibited the same dose dependency as the production of total inositol phosphates for each mitogen suggesting that the two responses might be mediated through identical receptors. Acute pre-treatment with TPA allowed the dissociation of inositol lipid hydrolysis from PtdCho breakdown, since it inhibited inositol phosphate accumulation but stimulated choline generation. The loss of mitogen stimulated choline release in cells pre-treated with the phorbol ester for 48 h was not due to loss of stimulated inositol phosphate production which was reproducibly enhanced in these 'down-regulated' cells.

Arginine Vasopressin↗
PubMed1989

High-performance liquid chromatographic determination of pirprofen and five of its metabolites in human plasma without hydrolysis and in human urine before and after chemical hydrolysis.

Selective high-performance liquid chromatographic methods for the simultaneous determination of pirprofen and five of its metabolites either in plasma or in urine before and after chemical hydrolysis were developed. After addition of an internal standard and a buffer, the compounds were extracted from plasma using reversed-phase C18 Bond-Elut columns and from urine using pre-packed silica Extrelut 1 columns, back-extraction into sodium hydroxide and acidification of the alkaline phase before injection. Pirprofen, its five metabolites and the internal standard were separated using a linear elution gradient chromatographic system and wavelength programming. The analysis of spiked samples demonstrated the good accuracy and precision of the methods with limits of quantitation of 100 or 200 ng/ml for the different compounds in plasma, 200 or 360 ng/ml in urine without hydrolysis and 1 or 1.8 micrograms/ml in urine after chemical hydrolysis.

Chemical Phenomena↗
PubMed2001

Correlation of base consumption with the degree of hydrolysis in enzymic protein hydrolysis.

It is fairly easy to control the enzymic hydrolysis of proteins in alkaline conditions by measuring the base consumption required to keep the pH constant in the reactor. Unfortunately, however, base consumption is not related in any simple way to the degree of hydrolysis reached at any given moment and to establish this relationship it is essential to find out the mean pK of the alpha-amino groups released during the hydrolytic process. We have shown here that the correct mean pK value varies according to the pH of the working conditions and that the relationship between these values may depend upon the kind of protein and protease used. We have put forward a method for determining this relationship experimentally by using a given protein-protease system, consisting of an alkaline titration of the raw protein and when partially hydrolysed. We have tested the results predicted by our theoretical model by applying it to the hydrolysis of whey proteins with a bacterial protease from Bacillus licheniformis at 50 degrees C, pH 8.0. This model can easily be applied to any hydrolytic process involving the appearance of functional groups that are partially protonizable under the working conditions in question in order to follow the kinetics of the reaction via the consumption of the neutralizing agent required to keep pH constant.

Animals↗
PubMed1981

Oxygen-18 leaving group kinetic isotope effects on the hydrolysis of nitrophenyl glycosides. 2. Lysozyme and beta-glucosidase: acid and alkaline hydrolysis.

Oxygen-18 leaving group kinetic isotope effects (KIEs) have been measured for a set of glycosyl transfer reactions with p-nitrophenyl beta-D-glycosides as substrates. Acid-catalyzed hydrolysis and alkaline hydrolysis exhibit KIEs of K16/k18 = 1.0355 +/- 0.0015 and 1.0386 +/- 0.0032, respectively. Lysozyme and beta-glucosidase A show KIEs on Vmax/Km (V/K) of (V/KI)16/(V/K)18 = 1.0467 +/- 0.0015 and 1.0377 +/0 0.0061, respectively. The large magnitude of these KIEs requires that carbon-oxygen bond scission be far advanced in the transition states for these reactions; therefore in the transition states for the first irreversible steps in these reaction sequences, scission of the glycosidic bond must be essentially complete for the reactions catalyzed by lysozyme and beta-glucosidase A, which are thought to proceed via SN1 and SN2 mechanisms, respectively. Acid-catalyzed hydrolysis is shown to proceed through a transition state involving at least 80% C-O bond cleavage and only partially proton transfer to the leaving p-nitrophenyl oxygen atom.

Glucosidases↗
PubMed2000

Paraoxon and parathion hydrolysis by aqueous molybdenocene dichloride (Cp2MoCl2): first reported pesticide hydrolysis by an organometallic complex.

We report the first case of an organometallic complex that effectively hydrolyzes the organophosphate pesticides parathion and paraoxon. The complex is the water-soluble compound bis(eta 5-cyclopentadienyl)molybdenum(IV) dichloride (1), which hydrolyzes parathion to produce ethanol and deethyl parathion in a biphasic reaction in D2O. Rate accelerations were 130 and 10(5) at pH 7 and 3, respectively. Paraoxon is readily hydrolyzed by 1 to yield p-nitrophenol and diethyl phosphate with rate accelerations of 2300 and 27 at pH 7 and 3, respectively. Kinetic data for paraoxon hydrolysis by 1 are consistent with a process that involves intermolecular (delta S++ = -49 +/- 10 eu) hydroxide attack on the phosphate triester in which the aquated 1 serves as a coordinated Lewis acid that activates the organophosphate. Interestingly parathion hydrolysis by 1 occurs via nucleophilic attack at the alpha-carbon of the phosphorothioate pesticide that involves C-O bond cleavage. These parathion results represent one of the few cases of this type of unusual hydrolytic chemistry and the first case of an organometallic complex that accelerates organophosphate pesticide hydrolysis.

Catalysis↗
PubMed1997

Controlled hydrolysis of ceftiofur sodium, a broad-spectrum cephalosporin; isolation and identification of hydrolysis products.

Ceftiofur sodium is the salt of (6R,7R)-7-{[(2-amino-4-thiazolyl)-Z-(methoxyimino)acetyl]amino}-3-{[(2-+ ++furanylcarbonyl)thio]methyl}-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2- ene-2-carboxylic acid. This compound is very susceptible to acid, alkaline-, and enzyme-catalyzed hydrolysis, producing a number of unstable degradation products. In this report, we describe the preparation and identification of the hydrolysis products that are formed under controlled alkaline conditions. The primary hydrolysis product was desfuroyl ceftiofur, which is the most abundant metabolite in bovine blood. Desfuroyl ceftiofur was carefully oxidized with H2O2 to prepare the disulfide dimer, a urinary metabolite of ceftiofur sodium in the rat and cattle. Under acidic conditions, desfuroyl ceftiofur was converted to the corresponding thiolactone. The preparation of desacetyl cefotaxime, which is the oxygen analog of desfuroyl ceftiofur, is also described. Furoic acid was readily formed by hydrolytic cleavage of the thioester bond. Thiofuroic acid, formed by the less common cleavage on the alkyl side of the thioester bond, was also isolated.

Cephalosporins↗
PubMed2006

Transition state effects in the acid-catalyzed hydrolysis of 5-methoxyacenaphthylene 1,2-oxide: implications for the mechanism of acid-catalyzed hydrolysis of cyclopenta[cd]pyrene 3,4-oxide.

5-Methoxyacenaphthylene 1,2-oxide (5) was synthesized by the reaction of 5-methoxyacenaphthylene with dimethyldioxirane. The rates and products from the acid-catalyzed and pH-independent reactions of 5 in 50:50 dioxane/water have been determined. The half-life of the pH-independent reaction of this very reactive epoxide in 50:50 dioxane/water is only 22 s. Acid-catalyzed hydrolysis of 5 in 50:50 dioxane/water yields 62% of cis diol 6, 37% of trans diol 7, and approximately 1% of 5-methoxy-1,2-dihydroacenaphthylen-1-one (8). The pH-independent reaction of 5 yields mostly ketone 8 (94%), along with minor amounts of cis and trans diols. The relative stabilities of cis and trans diols 6 and 7 were determined by treating either cis or trans diol with perchloric acid in water solutions and following the approach to an equilibrium cis/trans diol mixture as a function of time. At equilibrium, the ratio of cis and trans diols is 19:81, which establishes that trans diol 7 is more stable than cis diol 6. The acid-catalyzed hydrolysis of epoxide 5 therefore yields the less stable cis diol as the major product. It is concluded that transition state effects therefore selectively stabilize the transition state for attack of water on the intermediate carbocation leading to the less stable cis diol. These results suggest that transition state effects are also responsible for formation of the major cis diol in the acid-catalyzed hydrolysis of cyclopenta[cd]pyrene 3,4-oxide, which has a cyclopenta-fused ring similar to that in 5.

Acenaphthenes↗
PubMed1999

Nonenzymatic hydrolysis of oligoribonucleotides. V. The elements affecting the process of self-hydrolysis.

Chemical instability of some of the phosphodiester bonds, often observed in large RNAs, visualizes the autocatalytic properties of this class of nucleic acids. Unexpectedly, selective hydrolysis occurs also in short oligoribonucleotides (as short as a tetramer or hexamer). Herein, we describe additional experiments which support the conclusion that the hydrolysis is not due to ribonuclease contamination but is of autocatalytic origin and is related to the sequence and structure of single-stranded oligomers. Moreover, we show that the presence in the reaction mixture of polyamines, such as spermidine, is essential for hydrolysis of oligoribonucleotides.

Animals↗
PubMed2000

[Coupling of proteolysis with ATP hydrolysis by Escherichia coli Lon proteinase. I. Kinetic aspects of ATP hydrolysis].

Some aspects of the ATPase function of the Escherichia coli Lon protease were studied around the optimum pH value. It was revealed that, in the absence of the protein substrate, the maximum ATPase activity of the enzyme is observed at an equimolar ratio of ATP and Mg2+ ions in the area of their millimolar concentrations. Free components of the substrate complex (ATP-Mg)2- inhibit the enzyme ATPase activity. It is hypothesized that the effector activity of free Mg2+ ions is caused by the formation of the "ADP-Mg-form" of the ATPase centers. It was shown that the activation of ATP hydrolysis in the presence of the protein substrate is accompanied by an increase in the affinity of the (ATP-Mg)2- complex to the enzyme, by the elimination of the inhibiting action of free Mg2+ ions without altering the efficiency of catalysis of ATP hydrolysis (based on the kcat value), and by a change in the type of inhibition of ATP hydrolysis by the (ADP-Mg)- complex (without changing the Ki value). Interaction of the Lon protease protein substrate with the enzyme area located outside the peptide hydrolase center was demonstrated by a direct experiment.

ATP-Dependent Proteases↗
PubMed1985

Activator protein required for the enzymatic hydrolysis of cerebroside sulfate. Deficiency in urine of patients affected with cerebroside sulfatase activator deficiency and identity with activators for the enzymatic hydrolysis of GM1 ganglioside and globotriaosylceramide.

Urine specimens from two sibs affected with cerebroside sulfatase activator deficiency were examined to ascertain whether the deficiency of the supplementary activator protein required for the enzymatic hydrolysis of cerebroside sulfate was also evident in urine. Material from chromatographic fractionations was examined for the activator activity to avoid ambiguities resulting from protein inhibition. There were substantial deficits in all chromatographic fractions corresponding to activator-containing fractions of control urines. Since patient urines contained elevated amounts of lactosylceramide, digalactosylceramide, and globotriaosylceramide and since similarities between activators for cerebroside sulfate and GM1 ganglioside hydrolyses had been noted previously, the chromatographic fractions were also examined for activators in other glycosphingolipid hydrolase systems. There was coincidence of activators for the GM1 ganglioside/beta-galactosidase and the globotriaosylceramide/alpha-galactosidase A reactions with the cerebroside sulfatase activator in control urine fractions, and the patients' urines were deficient in activator activities for the three reactions. Identity of the three activators was suggested and antiserum to purified GM1 ganglioside activator was used to test this possibility. There were depressed levels of cross-reacting material in fractions of patient urines by Ouchterlony double diffusion and in unfractionated urine by enzyme-linked immunosorbent assay. Purified activators for the cerebroside sulfate and GM1 ganglioside systems showed lines of identity with no spurring on Ouchterlony double diffusion, identical mobility on immunoelectrophoresis, and similar stimulatory activities toward hydrolysis of the three glycosphingolipid species by their respective enzymes. Finally, the three activator activities were retained by anti-GM1-activator IgG coupled to Sepharose 4B. The results suggest strongly that the same protein entity serves as activator for the enzymatic hydrolysis of cerebroside sulfate, GM1 ganglioside, and globotriaosylceramide.

Animals↗
PubMedDate not supplied

[Hydrolysis of lorazepam with hydrochloric acid. Qualitative and quantitative analysis of byproducts of hydrolysis].

Results of the identification of some additional products in the hydrochloric acid hydrolysis of lorazepam are reported, together with some quantitative results about the hydrolysis products under varying circumstances. It appeared that the amount of byproducts was reduced by applying hydrochloric acid of good quality and further on hydrolysis time had to be short.

Chromatography, Gas↗
PubMed1996

Suggested Improvement in the Ing-Manske Procedure and Gabriel Synthesis of Primary Amines: Kinetic Study on Alkaline Hydrolysis of N-Phthaloylglycine and Acid Hydrolysis of N-(o-Carboxybenzoyl)glycine in Aqueous Organic Solvents.

A slight modification of the Gabriel synthesis of primary amines is suggested on the basis of the observed and reported values of rate constants for the alkaline and acid hydrolyses of phthalimide, phthalamic acid, benzamide, and their N-substituted derivatives. The suggested procedure requires shorter reactions time and milder acid-base reaction conditions compared with the conventional acid-base hydrolysis in the Gabriel synthesis. A slight modification in the Ing-Manske procedure is also suggested. Pseudo-first-order rate constants, k(obs), for hydrolysis of N-phthaloylglycine, NPG, decrease from 24.1 x 10(-3) to 7.72 x 10(-3) and 6.12 x 10(-3) s(-1) with increasing acetonitrile and 1,4-dioxan contents, respectively, from 2 to 50% v/v (all the percentages given in the paper are vol %), while increasing the organic cosolvents content from 50 to 80% increases k(obs) from 7.72 x 10(-3) to 19.7 x 10(-3) s(-1) for acetonitrile and from 6.12 x 10(-3) to 52.8 x 10(-3) s(-1) for 1,4-dioxan, in aqueous organic solvents containing 0.004 M NaOH at 35 degrees C. The rate constants for NPG hydrolysis decrease from 2.11 x 10(-2) to 1.19 x 10(-4) s(-1) with increasing MeOH content from 2 to 84%, in aqueous organic solvents containing 2% MeCN and 0.004 M NaOH at 35 degrees C.

Journal Article↗
PubMed1981

Hydrolysis of nucleoside triphosphates catalyzed by the recA protein of Escherichia coli. Hydrolysis of UTP.

Hydrolysis of UTP catalyzed by the recA protein of Escherichia coli is stimulated by both double- (DS) and single-stranded (SS) DNA. DS DNA-dependent UTPase activity has a sharp optimum near pH 6. SS DNA-dependent UTP hydrolysis also is optimal near pH 6, although considerable activity remains at pH 8. Both SS and DS DNA-dependent UTPase activities are nonlinearly dependent on recA protein concentration at pH 6 but the SS DNA-dependent reaction shows a linear dependence on enzyme concentration at pH 8. The Km for UTP in the SS DNA-dependent reaction decreases from 147 microM at pH 8 to 33 microM at pH 6. The Km for UTP in the DS DNA-dependent reaction is 247 microM at pH 6. In addition, the Hill coefficient for UTP in the SS DNA-dependent reaction decreases from 3.5 at pH 8 to 1.9 at pH 6, while in the DS DNA-dependent reaction, the Hill coefficient is 2.4 at pH 6. Thus, the UTPase activity of the recA protein differs from the ATPase activity of recA protein primarily in the pH dependence of KmUTP, Vmax, and response to enzyme concentration of the SS DNA-dependent hydrolysis reaction. These differences may be related to the inability of UTP to substitute effectively for ATP in recA protein-promoted annealing and assimilation of SS DNA.

Bacterial Proteins↗
PubMed1998

Nitrilase catalyzes amide hydrolysis as well as nitrile hydrolysis.

While amides were reported to be completely inert as substrates for all nitrilases reported to date, the nitrilase from Rhodococcus rhodochrous J1, which catalyzes the hydrolytic cleavage of the C-N triple bond in nitrile to form acid and ammonium, was surprisingly found to catalyze hydrolysis of amide to acid and ammonium stoichiometrically. This nitrilase exhibited a Km of 2.94 mM for benzamide, similar to that for benzonitrile as the original substrate (2.10 mM), but the Vmax for benzamide was six orders of magnitude lower than that for benzonitrile. Benzamide inhibited the nitrilase reaction in a reversible, apparently competitive manner. A mutant nitrilase containing alanine or serine instead of Cys165, which is essential for nitrilase catalytic activity, showed no amidase activity. This observation demonstrated that Cys165 plays a crucial role in the hydrolysis of amides as well as nitriles. Together with some reports that certain nitrilases were previously noted to produce low amounts of amide as a by-product from nitrile, the above unexpected findings suggested the existence of a common tetrahedral intermediate in the nitrilase reaction involving nitrile or amide as a substrate.

Aminohydrolases↗
PubMed1980

Phospholipase A2 inactivation of microsomal 17 beta-hydroxysteroid oxidoreductase: rates of phospholipid hydrolysis and enzyme inactivation, effects of hydrolysis products and properties of the phospholipase A2-treated enzyme.

Exposure of guinea pig liver microsomes to phospholipase A2 resulted in the nearly complete loss of 17 beta-hydroxysteroid oxidoreductase (17 beta-HSD) activity, the time course of which correlated with phospholipid hydrolysis and lysolecithin formation. Lysolecithin and unsaturated fatty acids added to microsomes also inactivated 17 beta-HSD indicating that they may contribute to the inactivation by phospholipase A2. If exposure to lysolecithin and fatty acids was minimized by including serum albumin in the reaction mixture, phospholipids were rapidly hydrolyzed; but in this case the extent of 17 beta-HSD inactivation was less and the rate of loss was significantly slower. The data suggest that phospholipid hydrolysis per se results in a destabilization of 17 beta-HSD resulting in the subsequent activity loss. The inactivation of 17 beta-HSD by lysolecithin and fatty acids has not been reported previously and is suggestive of a possible control mechanism in vivo.

17-Hydroxysteroid Dehydrogenases↗
PubMed1988

Hydrolysis of dansyl-peptide substrates by leucine aminopeptidase: origin of dansyl fluorescence changes during hydrolysis.

The origin of the fluorescence changes observed in stopped-flow experiments of the hydrolysis of three 5-(dimethylamino)naphthalene-1-sulfonyl-(dansyl) peptide substrates by porcine kidney cytosol leucine aminopeptidase has been investigated. The substrates used all have the potential to accept energy from aromatic residues of the enzyme via resonance energy transfer when they are bound as enzyme-substrate complexes, indicating that fluorescence changes due to the buildup and decay of such intermediates are possible. However, the fluorescence of these substrates differs from that of the products, and direct excitation of their dansyl groups during hydrolysis can also be responsible for the observed fluorescence changes due to changes in the concentrations of free substrate and product. The dansyl fluorescence changes observed with excitation wavelengths near 280 nm are not accompanied by quenching of the enzyme fluorescence, as would be expected if there were enzyme-to-substrate energy transfer. The magnitude of the maximal fluorescence change at a fixed concentration of substrate is also independent of the enzyme concentration. Furthermore, the excitation profile for the fluorescence changes shows that they arise from direct excitation of the dansyl group. Thus, there is no energy transfer in these reactions, and the fluorescence changes observed arise from direct excitation of the dansyl group and reflect the instantaneous concentration of substrate. This behavior contrasts sharply with that for the reaction of carboxypeptidase A with dansyl-Gly-Tyr, which has been studied as a positive control for an energy-transfer system.(ABSTRACT TRUNCATED AT 250 WORDS)

Carboxypeptidases↗
PubMed1981

Oxygen-18 leaving group kinetic isotope effects on the hydrolysis of nitrophenyl glycosides. 1. beta-galactosidease-catalyzed hydrolysis.

Oxygen-18 leaving group kinetic isotope effects (KIEs) have been determined on both Vmax (V) and Vmax/Km (V/K) for the beta-galactosidase-catalyzed hydrolysis of p-nitrophenyl beta-D-galactoside (I) and 2,4-dinitrophenyl beta-D-galactoside (II). The former substrate exhibits KIEs of 1.022 +/- 0.002 and 1.014 +/- 0.003 on V and V/K, respectively, while corresponding KIEs for the latter are 1.002 +/- 0.0009 and 1.030 +/- 0.003. These results indicate that bond scission is largely rate determining for I but not for II at substrate saturation. The first irreversible step for both substrates must involve cleavage of the bond to the nitrophenyl leaving group. The mechanism proposed for this reaction is characterized by two parallel pathways for substrate hydrolysis. The predominant route for all but the most reactive substrates involves a SN2 nucleophilic displacement of aglycon by the enzyme to yield a covalent galactosyl-enzyme which in turn is hydrolyzed via a nucleophilic attach by water. The most reactive substrates (e.g., II) from transiently an enzyme-bound galactosyl oxo-carbonium ion which partitions between enzyme to give the covalent galactosyl-enzyme and H2O to yield galactose.

Galactosidases↗