Use of stable isotopes in studies on the metabolism of amphetamine (1)

Microsomal coincubation of 1,1,1-²H₃-amphetamine and unlabelled N-hydroxyamphetamine yielded ²H-incorporation into recovered N-hydroxyamphetamine. The mole fraction of ²H in recovered phenylacetone was always close to but less than one, indicating that N-hydroxyamphetamine is not a necessary intermediate in the formation of phenylacetone. However, coincubation of ²H-labelled hydroxylamine with unlabelled 2-nitro-1-phenylpropane indicated an incorporation of ²H into both recovered nitro compound and phenylacetone. Some phenylacetone is thus formed from the nitro metabolite. Similar experiments showed phenylacetone oxime not to be a necessary intermediate in the conversion of hydroxylamine to the nitro compound. Incubation of phenyl-labelled (²H) phenylacetone gave 5 deuterium-labelled metabolites, including $\text{small}$ quantities of labelled benzoic acid, indicating that it is a true though minor metabolite.     

Human brain monoamine oxidase type B: mechanism of deamination as probed by steady-state methods

Recently, evidence has been published which suggests that [Husain, M., Edmondson, D. E., & Singer, T.P. (1982) Biochemistry 21, 595-600] monoamine oxidase [amine:oxygen oxidoreductase (MAO), EC 1.4.3.4] deaminates phenylethylamine and benzylamine via two distinct kinetic pathways which involve either binary or ternary complex formation, respectively. These conclusions were drawn largely from stopped-flow kinetic analysis performed on purified enzyme removed from its native membrane and in the presence of the inhibitory detergent Triton X-100. In this study, d-amphetamine and alternative substrates were used as steady-state probes of the kinetics of deamination by the B form of human brain MAO using native membrane-bound enzyme. Initial velocity studies showed mixed-type patterns for amphetamine inhibition of phenylethylamine, tryptamine, and tyramine when either amine or oxygen was the varied substrate. Slope and intercept vs. amphetamine concentration replots were linear in all cases except for phenylethylamine (hyperbolic); Ki values obtained from linear replots of slope or intercept values were comparable. In contrast, amphetamine was a competitive inhibitor of benzylamine deamination when amine concentration was varied and uncompetitive when oxygen concentration was varied; slope and intercept replots were linear for both. When benzylamine was the alternative substrate inhibitor and tyramine and tryptamine deamination was measured, mixed-type inhibition patterns were obtained when either amine or oxygen concentration was varied; replots of slope and intercept were linear in all cases.(ABSTRACT TRUNCATED AT 250 WORDS)     

18O studies of the peroxidase-catalyzed oxidation of N-methylcarbazole. Mechanisms of carbinolamine and carboxaldehyde formation

Chloroperoxidase, horseradish peroxidase, hemoglobin, myoglobin, lactoperoxidase, and microperoxidase catalyzed the ethyl hydroperoxide-dependent oxidation of N-methylcarbazole to N-(hydroxymethyl)carbazole and N-formylcarbazole as major products. Mass spectral analysis of the N-(hydroxymethyl)carbazole formed during the peroxidase-catalyzed N-demethylation of N-methylcarbazole in 18O-enriched medium indicated partial incorporation (7.5-25.9%) of solvent water oxygen into the carbinolamine intermediate in all systems investigated, suggesting that the peroxidase active site is partially accessible to solvent water during N-demethylation. In contrast, solvent water oxygen was not incorporated into the N-formylcarbazole formed during the peroxidase-catalyzed oxidation of N-methylcarbazole. N-(Hydroxymethyl)carbazole was not further metabolized by the peroxidases in the presence of ethyl hydroperoxide, indicating that it is not an intermediate in N-formylcarbazole formation. The horseradish peroxidase-catalyzed formation of N-formylcarbazole was decreased by 77% when the hydroperoxide-supported reactions were carried out in a nitrogen atmosphere, while the formation of N-(hydroxymethyl)carbazole was decreased by 46%. When the horseradish peroxidase-catalyzed reactions were carried out in a 18O2 atmosphere, 18O incorporation into N-(hydroxymethyl)carbazole was 64.4% of the total oxygen, while 81.8% of the oxygen incorporated into N-formylcarbazole came from 18O2. These results suggest that there are two different mechanisms for the formation of N-(hydroxymethyl)carbazole, both involving the initial oxidation of N-methylcarbazole to a neutral carbon-centered radical. The radical can be further oxidized in the enzyme active site to an iminium cation, which reacts with water derived from either the oxidant or the medium to form the carbinolamine. Alternatively, the substrate radical can react with molecular oxygen to form a hydroperoxy radical, which decomposes to form the carboxaldehyde and carbinolamine.     

Kinetic mechanism of phenylacetone monooxygenase from Thermobifida fusca

Phenylacetone monooxygenase (PAMO) from Thermobifida fusca is a FAD-containing Baeyer-Villiger monooxygenase (BVMO). To elucidate the mechanism of conversion of phenylacetone by PAMO, we have performed a detailed steady-state and pre-steady-state kinetic analysis. In the catalytic cycle ( k cat = 3.1 s (-1)), rapid binding of NADPH ( K d = 0.7 microM) is followed by a transfer of the 4( R)-hydride from NADPH to the FAD cofactor ( k red = 12 s (-1)). The reduced PAMO is rapidly oxygenated by molecular oxygen ( k ox = 870 mM (-1) s (-1)), yielding a C4a-peroxyflavin. The peroxyflavin enzyme intermediate reacts with phenylacetone to form benzylacetate ( k 1 = 73 s (-1)). This latter kinetic event leads to an enzyme intermediate which we could not unequivocally assign and may represent a Criegee intermediate or a C4a-hydroxyflavin form. The relatively slow decay (4.1 s (-1)) of this intermediate yields fully reoxidized PAMO and limits the turnover rate. NADP (+) release is relatively fast and represents the final step of the catalytic cycle. This study shows that kinetic behavior of PAMO is significantly different when compared with that of sequence-related monooxygenases, e.g., cyclohexanone monooxygenase and liver microsomal flavin-containing monooxygenase. Inspection of the crystal structure of PAMO has revealed that residue R337, which is conserved in other BVMOs, is positioned close to the flavin cofactor. The analyzed R337A and R337K mutant enzymes were still able to form and stabilize the C4a-peroxyflavin intermediate. The mutants were unable to convert either phenylacetone or benzyl methyl sulfide. This demonstrates that R337 is crucially involved in assisting PAMO-mediated Baeyer-Villiger and sulfoxidation reactions.     

Schiff bases or glycosylamines: crystal and molecular structures of four derivatives of D-mannose

Crystal and molecular structures of four derivatives of D-mannose are described. Each could exist as either an open-chain Schiff base or as a glycosylamine in the solid state. The derivative formed upon reaction of D-mannose with hydroxylamine is an open-chain oxime, but those formed upon reaction with semicarbazide, aniline, and p-chloroaniline are glycosylamines. The oxime, which crystallizes as the syn-(E) isomer, has a fully extended carbon chain. The glycosylamines are all beta-pyranoses. The packing arrangement of the oxime involves 'head-to-tail' hydrogen bonding. The semicarbazide derivative, which crystallizes as a dihydrate, features a hydrogen-bonded intramolecular bridge formed by the two water molecules and linking O-6 to the carbonyl oxygen atom. The packing arrangements of the aniline and p-chloroaniline derivatives differ from each other but are nevertheless closely related by similar hydrogen-bonding interactions.     

Density functional theory studies on hydroxylamine mechanism of cyclohexanone ammoximation on titanium silicalite-1 catalyst

The hydroxylamine mechanism of cyclohexanone ammoximation on defective titanium active site of titanium silicalite-1 (TS-1) was simulated using two-layer ONIOM (M062X/6-31G**:PM6) method. A new energy favorable reaction route was found, which contained two parts: (1) the catalytic oxidation of adsorbed NH₃ to form hydroxylamine using the Ti-OOH as an active oxidant formed by reacting H₂O₂ with the defective Ti active site; (2) the subsequent noncatalytic oximation of desorbed hydroxylamine and cyclohexanone out of TS-1 pores to form cyclohexanone oxime. In the catalytic formation of hydroxylamine on the Ti active site of TS-1, the proposed mechanism of two-step single-proton transfer aided by a lattice oxygen atom bonded to Ti atom need a lower reaction energy than the mechanism proposed before. In the subsequent noncatalytic oximation of hydroxylamine and cyclohexanone, which contained two elementary reaction steps in total, the mechanisms of one-step double-proton transfer in the first elementary reaction step and the subsequent one-step three-proton transfer for the second elementary reaction step were proposed, in which the solvent water molecules played a very important role in assisting and stabilizing the proton transfer processes.     

Effect of methylamine on the reaction of alpha 2-macroglobulin with enzymes.

The kinetics of reaction of alpha 2-macroglobulin (alpha 2M) with thrombin and with trypsin were studied in the presence and absence of methylamine. The rate of enzyme-induced thiol release was found to be the same whether or not amine was present. The result suggests that covalent bond formation and enzyme-catalyzed amine incorporation proceed via a common (enzyme-dependent) rate-determining step. The reaction of lysyl-modified enzymes (which show poor covalent binding with alpha 2M) was similarly unaffected by amine, indicating that enzyme-catalyzed steps were also rate determining for hydrolysis of the thiol ester. The products of the reactions were analyzed by native and denaturing gel electrophoresis. Methylamine did not affect the total binding of enzyme to alpha 2M but did cause a substantial decrease in covalent binding. Surprisingly, not all covalent complexes were affected by the presence of amine: complexes in which enzyme was covalently bound to one half-molecule increased compared to the reaction with no amine; complexes in which two half-molecules are cross-linked by two bonds to a single enzyme were substantially reduced, however. The results are consistent with a mechanism of reaction in which an enzyme-dependent step is rate determining. This step is accompanied by activation of two thiol esters. One of these reacts immediately with the bound enzyme (or may be hydrolyzed if the enzyme amine groups are blocked). The other activated center is capable of reaction with external nucleophiles such as methylamine.     

Ibogaine reduces amphetamine-induced locomotor stimulation in C57BL/6By mice, but stimulates locomotor activity in rats.

The effect of ibogaine hydrochloride on locomotor stimulation induced by d-amphetamine sulfate was tested in male C57BL/6By mice and in female Sprague-Dawley rats. In mice, locomotor stimulation induced by d-amphetamine at 1 or 5 mg/kg s.c. was reduced by prior administration of one or two injections of ibogaine (40 mg/kg), given 2 or 18 hours earlier. This reduction in locomotor activity persisted for two days. Locomotor stimulation induced by a higher dose (10 mg/kg) of d-amphetamine was not reduced by such prior administration of ibogaine. A lower dose of ibogaine (20 mg/kg) did not reduce the subsequent locomotor activity induced by d-amphetamine. Ibogaine decreased striatal dopamine levels, while d-amphetamine increased them. Ibogaine treatment (2 x 40 mg/kg, 18 hours apart) induced a decrease by 30% in the level of striatal dopamine and its metabolites measured in tissue extracts 3 hours after the second ibogaine injection. One hour after d-amphetamine (5 mg/kg) administration, the level of striatal dopamine increased by 26%. Although the level of striatal dopamine was initially lower in the ibogaine-pretreated mice, d-amphetamine (5 mg/kg) administration induced an increase in striatal dopamine and its metabolites. The effect of ibogaine seems to be species specific, since in rats pretreated with ibogaine 18 hours before d-amphetamine, locomotor stimulation induced by d-amphetamine was further increased. In addition, the in vitro electrical-evoked release of [3H]dopamine from striatal tissue was either unchanged or inhibited in the presence of d-amphetamine, and after ibogaine pretreatment in vivo, the release of tritium in the presence of d-amphetamine was inhibited or stimulated in mice and rats, respectively.     

Studies of the mechanism of glutamine synthetase utilizing pH-dependent behavior in catalysis and binding

The pKa values of enzyme groups of Escherichia coli glutamine synthetase which affect catalysis and/or substrate binding were determined by measuring the pH dependence of Vmax and V/K. Analysis of these data revealed that two enzyme groups are required for catalysis with apparent pKa values of approximately 7.1 and 8.2. The binding of ATP is essentially independent of pH in the range studied while the substrate ammonia must be deprotonated for the catalytic reaction. Using methylamine and hydroxylamine in place of ammonia, the pKa value of the deprotonated amine substrate as expressed in the V/K profiles was shifted to a lower pKa value for hydroxylamine and a higher pKa value for methylamine. These data indicate that the amine substrate must be deprotonated for binding. Hydroxylamine is at least as good a substrate as ammonia judged by the kinetic parameters whereas methylamine is a poor substrate as expressed in both the V and V/K values. Glutamate binding was determined by monitoring fluorescence changes of the enzyme and the data indicate that a protonated residue (pKa = 8.3 +/- 0.2) is required for glutamate binding. Chemical modification by reductive methylation with HCHO indicated that the group involved in glutamate binding most likely is a lysine residue. In addition, the Ki value for the transition state analog, L-3-amino-3-carboxy-propanesulfonamide was measured as a function of pH and the results indicate that an enzyme residue must be protonated (pKa = 8.2 +/- 0.1) to assist in binding. A mechanism for the reaction catalyzed by glutamine synthetase is proposed from the kinetic data acquired herein. A salt bridge is formed between the gamma-phosphate group of ATP and an enzyme group prior to attack by the gamma-carboxyl of glutamate on ATP to form gamma-glutamyl phosphate. The amine substrate subsequently attacks gamma-glutamyl phosphate resulting in formation of the tetrahedral adduct before phosphate release. A base on the enzyme assists in the deprotonation of ammonia during its attack on gamma-glutamyl phosphate or after the protonated carbinol amine is formed. Based on the kinetic data with the three amine substrates, catalysis is not rate-limiting through the pH range 6-9.     

Preparation and antigenic properties of 5alpha-dihydrotestosterone-11-(O-carboxymethyl) oxime-BSA conjugate.

The C-11 (O-carboxymethyl) oxime derivative of 5-alphadihydrotestosterone (5alphaDHT) has been prepared. Due to steric hindrance at C-11, a novel two step procedure was used to introduce the (O-carboxymethyl) oxime at this position. Condensation of this oxime to bovine serum albumin afforded a conjugate which produced anti-5alphaDHT sera inoculated rabbits. Apart from a 30% cross reaction with testosterone, the antisera was reasonably specific for 5alphaDHT.     

Mechanism of oxygen exchange in actin-activated hydrolysis of adenosine triphosphate by myosin subfragment 1.

The gamma-phosphoryl groups of two intermediates (M-ATP and M-ADP-P1) in the pathway of MgATP hydrolysis by myosin undergo extensive oxygen exchange with water. Actin activates the overall rate of hydrolysis at a rate-limiting step which follows these exchange reactions. Thus, actin, by decreasing the turnover time of hydrolysis, would be expected to proportionately decrease the time available for oxygen exchange. Using subfragment 1 of myosin, the turnover time of hydrolysis can be varied over a wide range by changing the concentration of actin. An estimate for the rate constant of exchange can then be obtained by relating these turnover times to measured values for oxygen exchange (incorporation of 18O from H218O into the inorganic phosphate (Pi) released by hydrolysis). The results of such an experiment, with turnover times between 0.2 and 25 s, indicate that, for each gamma-phosphoryl group, one oxygen from the medium is added rapidly (to cleave the phosphoryl group or form a pentacoordinate phosphroyl complex); two more oxygens exchange with a rate constant, kc, of about 1 s-1; and a fourth oxygen exchanges slowly with ke about 0.2 s-1. The higher value is about 18 times smaller than the rate constant, 5-3, for the reverse cleavage step of the myosin pathway, which is postulated to be responsible for oxygen exchange. The data, then, indicate that the rate-limiting step for oxygen exchange is not k-3, but may be the rate of rotation of oxygens around the phosphorus atom, with one oxygen severely restricted by its binding to the active site. The finding that kc differs for the four oxygens in each phosphate group is related to past observations on myosin-catalyzed oxygen exchange.     

Formation and structure of Cu(II)-poly(L-lysine) complexes in aqueous solution.

Cu(II)-poly(L-lysine) complexes have been studied using potentiometric titrations, optical absorption and circular dichroism spectra. As in the Cu(II)-poly(L-arginine) system studied previously potentiometric and spectral data consistently show that two types of complexes are formed. The first formed below pH 7.6 contains two amine nitrogens and two oxygen from water molecules at the corners of a square in which the metal occupies the center. The second is obtained at pH above 7.6 when the oxygen atoms are replaced by two adjacent peptide nitrogens.     

Assessment of radiation-induced DNA damage caused by the incorporation of 99mTc-radiopharmaceuticals in murine lymphocytes using single cell gel electrophoresis

The DNA damage induced by the 99mTc-radiopharmaceuticals incorporation to the cell was determined by the single-cell gel electrophoresis in murine lymphocytes in vitro. The 99mTc-hexamethyl-propylene amine oxime (99mTc-HMPAO) and 99mTc-2, 5-dihydroxybenzoic acid (99mTc-gentisic acid) induced nearly 100% of cells with breaks and/or alkali labile sites, which is explained by the action of the Auger electrons produced by the decay of the 99mTc. These results agree with the doses of 1.6 and 1.0 Gy estimated by subcellular dosimetry for 99mTc-HMPAO that is incorporated in the cytoplasm, and the 99mTc-gentisic acid, which remains bonded to the cell membrane, respectively. The results imply that Auger electrons are able to cause important DNA damage, when the radionuclide is incorporated in the range of a few microns from the nuclei.     

Synthesis and analytical investigation of C‐terminally modified peptide aldehydes and ketone: application to oxime ligation

C‐terminally modified peptides aldehyde (glycinal and alpha‐oxo aldehyde peptides) and ketone (pyruvic acid‐containing peptide) were synthesised to get new insights into the mechanism of acido‐catalysed oxime ligation. Their tetrahedral hydrated forms were investigated in solution and in the gas phase, using NMR and in‐source collision‐induced dissociation mass spectrometry, respectively, and the kinetics of the oximation reactions followed using analytical HPLC. The results obtained confirmed that the first step of the oximation reaction was the limiting step for the pyruvic acid‐containing peptides because of the steric effect and of the carbon angular strain of the ketone. The second step is the determining step for the aldehyde peptides because the basicity of the oxygen of the hydroxyl function of the tetrahedral form is greater for glycinal than for alpha‐oxo aldehyde. These data strongly suggest that the hydrated form of the aldehyde partner has to be considered when oxime reactions are performed in aqueous buffer. Copyright © 2011 European Peptide Society and John Wiley & Sons, Ltd.     

Synthesis and DNA cleavage studies of novel quinoline oxime esters

New 2-chloro-3-formyl quinoline oxime esters were synthesized by the reaction of 2-chloro-3-formyl quinoline oximes with various benzoyl chlorides in the presence of triethyl amine and dichloromethane at 0°C. The DNA photo cleavage studies of some new oxime esters were investigated by neutral agarose gel electrophoresis at different concentrations (40μM and 80μM). Analysis of the cleavage products in agarose gel indicated that few of quinoline oxime esters (3d-i) converted into supercoiled pUC19 plasmid DNA to its nicked or linear form.     

The biosynthesis of cyanogenic glucosides in higher plants. Identification of three hydroxylation steps in the biosynthesis of dhurrin in Sorghum bicolor (L.) Moench and the involvement of 1-ACI-nitro-2-(p-hydroxyphenyl)ethane as an intermediate

N-Hydroxytyrosine, (E)- and (Z)-p-hydroxyphenyl-acetaldehyde oxime, p-hydroxyphenylacetonitrile, and p-hydroxymandelonitrile are established intermediates in the biosynthesis of the tyrosine-derived cyanogenic glucoside dhurrin (Halkier, B. A., Olsen, C. E., and M?ller, B. L. (1989) J. Biol. Chem. 264, 19487-19494. Simultaneous measurements of oxygen consumption and biosynthetic activity using a microsomal enzyme system isolated from etiolated sorghum seedlings demonstrate a requirement for three oxygen molecules in the conversion of tyrosine to p-hydroxymandelonitrile. Two oxygen molecules are consumed in the conversion of tyrosine to (E)-p-hydroxyphenylacetaldehyde oxime, indicating the existence of a previously undetected hydroxylation step in addition to that resulting in the formation of N-hydroxytyrosine. Radioactively labeled 1-nitro-2-(p-hydroxyphenyl)ethane was chemically synthesized and tested as a possible intermediate. Biosynthetic experiments demonstrate that the microsomal enzyme system metabolizes the nitro compound to the subsequent intermediates in dhurrin synthesis (Km = 0.05 mM; Vmax = 14 nmol/mg of protein/h). Low amounts of 1-nitro-2-(p-hydroxyphenyl)ethane are produced in the microsomal reaction mixtures when tyrosine is used as substrate. These data support the involvement of 1-nitro-2-(p-hydroxyphenyl)ethane or more likely its aci-nitro tautomer as an intermediate between N-hydroxytyrosine and p-hydroxyphenylacetaldehyde oxime. The conversion of (E)-p-hydroxyphenylacetaldehydeoxime to p-hydroxymandelonitrile requires a single oxygen molecule. The oxygen molecule is utilized for hydroxylation of p-hydroxyphenylacetonitrile into p-hydroxymandelonitrile. This indicates that the conversion of p-hydroxyphenylacetaldehyde oxime into p-hydroxyphenylacetonitrile proceeds by a simple dehydration reaction.     

IN VIVO INHIBITION OF RAT BRAIN PROTEIN SYNTHESIS BY d-AMPHETAMINE

Abstract— Between 1 and 4 h after rats received a single injection of d-amphetamine (15 mg/kg)(when brain polysomes are known to be disaggregated), the in vivo incorporation of [¹⁴C]lysine into trichloroacetic acid-precipitable brain protein was reduced by 28–48%. Incorporation of the ¹⁴C label into the protein present in a 100,000 g supernatant extract of whole brain was similarly reduced (by 44%). Amphetamine administration suppressed protein synthesis in rat cerebral cortex, cerebellum, hypothalamus, striatum, and brainstem to an equivalent extent. The drug did not significantly affect lysine pool sizes measured in these brain regions; thus the reduced incorporation of labeled lysine was not the result of an isotope dilution effect. We therefore conclude that the brain polysome disaggregation resulting from amphetamine administration is associated with decreased in vivo synthesis of some brain proteins.     

The influence of fish size size on the avoidance of hypoxia and oxygen selection by largemouth bass

When an oxygen gradient ranging from c . 10 to 95% air saturation was formed in a 5 m chamber, largemouth bass Micropterus salmoides avoided water in which dissolved oxygen values were <27% air saturation. There was a significant ( P <0·05) correlation between fish mass and the level of dissolved aquatic oxygen that was selected. Small fish (23–500 g) utilized waters of lower oxygen levels than did the larger fish (1000–3000 g). The results of this study suggest that largemouth bass are able to sense and avoid hypoxic water, and select aquatic oxygen tensions that maintain their metabolic scope for growth and activity.     

Exploring the structural basis of substrate preferences in Baeyer-Villiger monooxygenases: insight from steroid monooxygenase

Steroid monooxygenase (STMO) from Rhodococcus rhodochrous catalyzes the Baeyer-Villiger conversion of progesterone into progesterone acetate using FAD as prosthetic group and NADPH as reducing cofactor. The enzyme shares high sequence similarity with well characterized Baeyer-Villiger monooxygenases, including phenylacetone monooxygenase and cyclohexanone monooxygenase. The comparative biochemical and structural analysis of STMO can be particularly insightful with regard to the understanding of the substrate-specificity properties of Baeyer-Villiger monooxygenases that are emerging as promising tools in biocatalytic applications and as targets for prodrug activation. The crystal structures of STMO in the native, NADP(+)-bound, and two mutant forms reveal structural details on this microbial steroid-degrading enzyme. The binding of the nicotinamide ring of NADP(+) is shifted with respect to the flavin compared with that observed in other monooxygenases of the same class. This finding fully supports the idea that NADP(H) adopts various positions during the catalytic cycle to perform its multiple functions in catalysis. The active site closely resembles that of phenylacetone monooxygenase. This observation led us to discover that STMO is capable of acting also on phenylacetone, which implies an impressive level of substrate promiscuity. The investigation of six mutants that target residues on the surface of the substrate-binding site reveals that enzymatic conversions of both progesterone and phenylacetone are largely insensitive to relatively drastic amino acid changes, with some mutants even displaying enhanced activity on progesterone. These features possibly reflect the fact that these enzymes are continuously evolving to acquire new activities, depending on the emerging availabilities of new compounds in the living environment.