Stereoselectivity of phosphotriesterase with paraoxon derivatives: a computational study

The bacterial enzyme phosphotriesterase (PTE) exhibits stereoselectivity toward hydrolysis of chiral substrates with a preference for the S_p enantiomer. In this work, docking analysis and two explicit-solvent molecular dynamics (MD) simulations were performed to characterize and differentiate the structural dynamics of PTE bound to the S_p and R_p paraoxon derivative enantiomers (R_p-1 and S_p-1) hydrolyzed with distinct catalytic efficiencies. Comparative analysis of the molecular trajectories for PTE bound to R_p-1 and S_p-1 suggested that substrate binding induced conformational changes in the loops near the active site. After 100 ns of MD simulation, the Zn β²⁺ metal ion formed hexacoordinated- and tetracoordinated geometries in the S_p-1-PTE and R_p-1-PTE ensembles, respectively. Simulation results further showed that the hydrogen bond between Asp301 and His254 occurred with a higher probability after S_p-1 binding to PTE (47.5%) than that after R_p-1 binding (22.2%). These results provide a qualitative and molecular-level explanation for the 10 orders of magnitude increase in the catalytic efficiency of PTE toward the S_p enantiomer of paraoxon.     

The impact of β‐azido(or 1‐piperidinyl)methylamino acids in position 2 or 3 on biological activity and conformation of dermorphin analogues

The synthesis of new dermorphin analogues is described. The (R)‐alanine or phenylalanine residues of natural dermorphin were substituted by the corresponding α‐methyl‐β‐azidoalanine or α‐benzyl‐β‐azido(1‐piperidinyl)alanine residues. The potency and selectivity of the new analogues were evaluated by a competitive receptor binding assay in rat brain using [³H]DAMGO (a μ ligand) and [³H]DELT (a δ ligand). The most active analogue in this series, Tyr‐(R)‐Ala‐(R)‐α‐benzyl‐β‐azidoAla‐Gly‐Tyr‐Pro‐Ser‐NH₂ and its epimer were analysed by ¹H and ¹³C NMR spectroscopy and restrained molecular dynamics simulations. The dominant conformation of the investigated peptides depended on the absolute configuration around C^α in the α‐benzyl‐β‐azidoAla residue in position 3. The (R) configuration led to the formation of a type I β‐turn, whilst switching to the (S) configuration gave rise to an inverse β‐turn of type I′, followed by the formation of a very short β‐sheet. The selectivity of Tyr‐(R)‐Ala‐(R) and (S)‐α‐benzyl‐β‐azidoAla‐Gly‐Tyr‐Pro‐Ser‐NH₂ was shown to be very similar; nevertheless, the two analogues exhibited different conformational preferences. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.     

Stereoselective determination of apomorphine enantiomers in serum with a cellulose-based high-performance liquid chromatographic chiral column using solid-phase extraction and ultraviolet detection

A novel and rapid method for the separation and determination of R-(−)- and S-(+)-enantiomers of apomorphine in serum by high-performance liquid chromatography with UV detection is reported. The method involved a solid-phase extraction of the R-(−)- and S-(+)-enantiomers of apomorphine and the internal standard R-(−)-propylnorapomorphine from serum using a C₈ Bond-Elut column. The HPLC system consisted of a reversed-phase cellulose-based chiral column (Chiralcel OD-R, 250×4.6 mm I.D.) with a mobile phase of 35:65 (v/v) acetonitrile-0.05 M sodium perchlorate (pH 2.0, adjusted with 60–62% perchloric acid) at a flow-rate of 0.5 ml/min with UV detection at 273 nm. The detection and quantitation limits were 10 ng/ml for each enantiomer using 1 ml of serum. Linear calibration curves from 10 to 1000 ng/ml for both R-(−)- and S-(+)-enantiomers show coefficient of determination of more than 0.9995. Precision calculated as %R.S.D. and accuracy calculated as % error were 0.2–4.7 and 3.1–6.9%, respectively, for the R-(−)-enantiomer and 1.3–4.2 and 0.3–6.8%, respectively, for the S-(+)-enantiomer.     

Purification and Characterization of a Novel Alcohol Dehydrogenase from Leifsonia sp. Strain S749: a Promising Biocatalyst for an Asymmetric Hydrogen Transfer Bioreduction

To find microorganisms that could reduce phenyl trifluoromethyl ketone (PTK) to (S)-1-phenyltrifluoroethanol [(S)-PTE], styrene-assimilating bacteria (ca. 900 strains) isolated from soil samples were screened. We found that Leifsonia sp. strain S749 was the most suitable strain for the conversion of PTK to (S)-PTE in the presence of 2-propanol as a hydrogen donor. The enzyme corresponding to the reaction was purified homogeneity, characterized and designated Leifsonia alcohol dehydrogenase (LSADH). The purified enzyme had a molecular weight of 110,000 and was composed of four identical subunits (molecular weight, 26,000). LSADH required NADH as a cofactor, showed little activity with NADPH, and reduced a wide variety of aldehydes and ketones. LSADH catalyzed the enantioselective reduction of some ketones with high enantiomeric excesses (e.e.): PTK to (S)-PTE (>99% e.e.), acetophenone to (R)-1-phenylethanol (99% e.e.), and 2-heptanone to (R)-2-heptanol (>99% e.e.) in the presence of 2-propanol without an additional NADH regeneration system. Therefore, it would be a useful biocatalyst.     

Stereoselective high-performance liquid chromatographic analysis of ketoprofen and its acyl glucuronides in chronic renal insufficiency

A rapid, sensitive method was developed for the quantification of the R- and S-enantiomers of ketoprofen and their acyl glucuronide conjugates in the plasma and dialysate of hemodialysis-dependent anephric patients. Unconjugated R- and S-ketoprofen plasma concentrations were determined directly by liquid chromatography using a S,S-Whelk-O1 chiral stationary phase. R- and S-Ketoprofen glucuronide for use as standards were resolved using a C₁₈ reversed-phase HPLC column with a mobile phase containing the ion-pair reagent tetrabutylammonium hydrogen sulfate. Plasma glucuronides, however, could not be directly quantified due to matrix interference. Therefore, the glucuronides were isolated using reversed-phase HPLC and quantified after alkaline hydrolysis using the S,S-Whelk-O1 chiral stationary phase column.     

Protein recovery by continuous flotation

Summary Bovine serum albumin (BSA) was recovered from aqueous solutions by foam flotation. The protein concentrations in foam liquid C _S, in feed C _Pand in residue C _Rwere determined. The protein enrichment C _S/C_Pand the separation C _S/C_Ras well as the protein fraction in the foam liquid % BSA and foam liquid volume flow were determined as functions of the medium properties, operational conditions, and equipment parameters as well as concentrations of solid particles. At low protein concentrations in feed (e.g., C _P=40 mg · l^-1), and at 40° C, high performance was attained (C _X=2,000 mg · l^-1, C _R=4.4 mg · l^-1, C _S/C_P=50, C _S/C_R=450, 90% BSA. Continuous foam flotation is an efficient procedure for the recovery of low concentrations of proteins from liquid cultures.Abbreviations BSA bovine serum albumine - C _P protein concentration in feed (mg · l^-1) - C _R protein concentration in residue (mg · l^-1) - C _S protein concentration in foam liquid (mg · l^-1) - C _S/C_R protein separation (-) - C _S/C_P protein enrichment (-) - V _P feed rate (ml · min^-1) - V _R residue flow rate (ml · min^-1) - V _S foam liquid volume flow (ml · min^-1) - N number of theoretical stages in an ideal cascade (-) - temperature (° C) - mean residence time (min)     

Update on biochemical properties of recombinant Pseudomonas diminuta phosphotriesterase

Phosphotriesterase from Pseudomonas diminuta (PTE; EC 3.1.8.1) hydrolyzes organophosphate insecticides and chemical warfare agents. The two zinc cations in the active center can be substituted. Co²⁺-containing PTE is the most efficient but least stable isoform. Gel filtration showed that PTE is monomeric at the submicromolar concentrations used in kinetic assays. The analysis of the recombinant enzyme by X-ray fluorescence spectrometry and CCT-ICP-MS, confirms that recombinant Zn-PTE contains only Zn²⁺ whereas Co-PTE has Zn²⁺ and Co²⁺ in equimolar amount, with Co²⁺ most likely in the reported labile β-site. We noted that recombinant PTE is unstable at low concentrations and must be stabilized by a protein environment. We tested the effect of excess of various metal cofactors on PTE-catalyzed hydrolysis of paraoxon. We notably observed that ZnCl₂ induces a non-competitive partial inhibition of Zn²⁺- and Co²⁺-PTE at pH 8.5 (apparent Ki=155 μM and 52 μM, respectively). Inhibition results from interactions with colloidal Zn(OH)₂ formed in alkaline buffer that alters the catalytic machinery. NiCl₂ caused a similar effect at higher concentrations (apparent Ki=3 mM). We observed that mutating His123, a surface residue close to an alleged allosteric site, dramatically altered the bacterial expression yield of Co²⁺-PTE, Ki for Zn(OH)₂ inhibition, k_cat (up to 60 fold) for paraoxon hydrolysis, but not K_M. Issues addressed in this work are important for future biotechnological developments of PTE as a detoxifying enzyme.     

Enantioselectivity of aromatase inhibitors: Substituted 3-(4-aminophenyl)pyrrolidine-2,5-diones

The (+)-, (?)-, and (±)-forms of 1- and 1,3-substituted 3-(4-aminophenyl)pyrrolidine-2,5-dione have been examined as inhibitors of P450_AROM and P450_CSCC. The inhibitory potency for P450_AROM resided in the (+)-enantiomers of ( 1 ), ( 2 ), and ( 4 ) and the (?)-enantiomers of ( 3 ) and ( 5 ). These findings have been accommodated within a molecular graphics-derived model for binding of P450_AROM inhibitors to the substrate binding site. Crystallography showed that (+)-( 2 ) has the (R)-configuration. Spectral binding studies with human placental P450_AROM showed type II binding but although the K_S values were in line with the IC₅₀ values for individual compounds there was no overall correlation between K_S and IC₅₀ within the series. There was little difference in the inhibitory potency of the enantiomers and racemate of individual compounds toward P450_CSCC. © 1995 Wiley-Liss, Inc.     

Preferential catabolism of the (S)-enantiomer of the herbicide napropamide mediated by the enantioselective amidohydrolase SnaH and the dioxygenase Snpd in Sphingobium sp. strain B2

The (R)- and (S)-enantiomers of the chiral herbicide napropamide (NAP) show different biological activities and ecotoxicities. These two enantiomers behave differently in the environment due to enantioselective catabolism by microorganisms. However, the molecular mechanisms underlying this enantioselective catabolism remain largely unknown. In this study, the genes (snaH and snpd) involved in the catabolism of NAP were cloned from Sphingobium sp. B2, which was capable of catabolizing both NAP enantiomers. Compared with (R)-NAP, (S)-NAP was much more rapidly transformed by the amidase SnaH, which initially cleaved the amide bonds of (S)/(R)-NAP to form (S)/(R)-2-(1-naphthalenyloxy)-propanoic acid [(S)/(R)-NP] and diethylamine. The α-ketoglutarate-dependent dioxygenase Snpd, showing strict stereoselectivity for (S)-NP, further transformed (S)-NP to 1-naphthol and pyruvate. Molecular docking and site-directed mutagenesis analyses revealed that when the (S)-enantiomers of NAP and NP occupied the active sites, the distance between the ligand molecule and the coordination atom was shorter than that when the (R)-enantiomers occupied the active sites, which facilitated formation of the transition state complex. This study enhances our understanding of the preferential catabolism of the (S)-enantiomer of NAP on the molecular level.     

Constrained C-terminal hexapeptide neurotensin analogues containing a 3-oxoindolizidine skeleton

Summary In order to enforce different spatial orientations in the C-terminal hexapeptide of neurotensin (NT_8–13) and to gain information about the importance of the 10–11 peptide bond for binding to NT receptors, the Pro¹⁰-Tyr¹¹ fragment has been replaced with (2R,8S,8aR)-, (2S,8S,8aR)-, (2S,8S,8aS)-, (2S,8R,8aS)- and (2R,8R,8aS)-8-amino-2-benzyl-3-oxoindolizidine-2-carboxylic acid. Molecular dynamics calculations and energy minimization studies have shown that, contrarily to the Pro-Tyr moiety, none of these indolizidines display a tendency to adopt type I and III -turns, but those having (8S,8aR) or (8R,8aS) stereochemistry essentially adopt extended conformations and the (8S,8aS) stereoisomer prefers a nonstandard folding. The four diastereomeric NT_8–13 analogues incorporating (8S,8aR) or (8R,8aS) indolizidines displayed binding affinities for the brain NT receptor similar to that of [Ala¹¹]-NT_8–13 and only five- to ninefold lower than that of the corresponding analogue, [Phe¹¹]NT_8–13. Although this slight decrease could be attributed to differences in conformational behavior between these constrained NT_8–13 analogues and [Phe¹¹]NT_8–13 or NT_8–13, it is not clear whether the -turn around Pro¹⁰-AA¹¹ (AA=Phe, Tyr) is conserved upon receptor binding. An excessive restriction in the motions of the aromatic side chain, imposed by the highly steric constraint of the indolizidine moiety, emerges as an alternative explanation. The findings reported here demonstrate the possibility of replacing the Pro¹⁰-Tyr¹¹ dipeptide in NT_8–13 with a non-peptide residue without affecting considerably the affinity for brain NT receptors.     

Pre-steady-state kinetic and mutational insights into mechanisms of endo- and exonuclease DNA processing by mutant forms of human AP endonuclease

Human apurinic/apyrimidinic endonuclease APE1 catalyzes endonucleolytic hydrolysis of phosphodiester bonds on the 5′ side of structurally unrelated damaged nucleotides in DNA or native nucleotides in RNA. APE1 additionally possesses 3′-5′-exonuclease, 3′-phosphodiesterase, and 3′-phosphatase activities. According to structural data, endo- and exonucleolytic cleavage of DNA is executed in different complexes when the excised residue is everted from the duplex or placed within the intrahelical DNA cavity without nucleotide flipping. In this study, we investigated the functions of residues Arg177, Arg181, Tyr171 and His309 in the APE1 endo- and exonucleolytic reactions. The interaction between residues Arg177 and Met270, which was hypothesized recently to be a switch for endo- and exonucleolytic catalytic mode regulation, was verified by pre–steady-state kinetic analysis of the R177A APE1 mutant. The function of another DNA-binding–site residue, Arg181, was analyzed too; it changed its conformation when enzyme–substrate and enzyme–product complexes were compared. Mutation R181A significantly facilitated the product dissociation stage and only weakly affected DNA-binding affinity. Moreover, R181A reduced the catalytic rate constant severalfold due to a loss of contact with a phosphate group. Finally, the protonation/deprotonation state of residues Tyr171 and His309 in the catalytic reaction was verified by their substitution. Mutations Y171F and H309A inhibited the chemical step of the AP endonucleolytic reaction by several orders of magnitude with retention of capacity for (2R,3S)-2-(hydroxymethyl)-3-hydroxytetrahydrofuran-containing-DNA binding and without changes in the pH dependence profile of AP endonuclease activity, indicating that deprotonation of these residues is likely not important for the catalytic reaction.     

Structural Basis of Substrate Specificity of Plant 12-Oxophytodienoate Reductases

12-Oxophytodienoate reductase 3 (OPR3) is a FMN-dependent oxidoreductase that catalyzes the reduction of the cyclopentenone (9S,13S)-12-oxophytodienoate [(9S,13S)-OPDA] to the corresponding cyclopentanone in the biosynthesis of the plant hormone jasmonic acid. In vitro, however, OPR3 reduces the jasmonic acid precursor (9S,13S)-OPDA as well as the enantiomeric (9R,13R)-OPDA, while its isozyme OPR1 is highly selective, accepting only (9R,13R)-OPDA as a substrate. To uncover the molecular determinants of this remarkable enantioselectivity, we determined the crystal structures of OPR1 and OPR3 in complex with the ligand p-hydroxybenzaldehyde. Structural comparison with the OPR1:(9R,13R)-OPDA complex and further biochemical and mutational analyses revealed that two active-site residues, Tyr78 and Tyr246 in OPR1 and Phe74 and His244 in OPR3, are critical for substrate filtering. The relatively smaller OPR3 residues allow formation of a wider substrate binding pocket that is less enantio-restrictive. Substitution of Phe74 and His244 by the corresponding OPR1 tyrosines resulted in an OPR3 mutant showing enhanced, OPR1-like substrate selectivity. Moreover, sequence analysis of the OPR family supports the filtering function of Tyr78 and Tyr246 and allows predictions with respect to substrate specificity and biological function of thus far uncharacterized OPR isozymes. The discovered structural features may also be relevant for other stereoselective proteins and guide the rational design of stereospecific enzymes for biotechnological applications.     

Enantioselectivity and stereoselectivity in the reactions of the enantiomers of the platinum complex [PtCl2(ahaz)] (ahaz = 3(R)- or 3(S)-aminohexahydroazepine) with DNA

The platinum-DNA adduct profile formed by the R- and S-enantiomers of [PtCl₂(ahaz)] (ahaz = 3(R)-aminohexahydroazepine or 3(S)-aminohexahydroazepine) on reaction with salmon sperm DNA were characterised using HPLC and GFAAS (graphite furnace atomic absorption spectrometry) analyses. At a platinum to nucleotide ratio (R_t) equalling 0.05, the R-enantiomer forms a substantially larger amount (approximately 60%) of monofunctional adducts than the S-enantiomer (less than 35%). Fewer intrastrand GpG adducts are formed by the R-enantiomer (approximately 21%) than the S-enantiomer (approximately 37%). For both enantiomers, two isomeric GpG adducts, corresponding to the different orientations of the primary amine of ahaz ligand with respect to the O6 atom of the 5′ guanine, were observed in the ratios of 1:1.3 and 1:4.3 for the R- and S-enantiomers, respectively. The reasons for this enantioselectivity and stereoselectivity are discussed.     

Evaluation of enantioselectivity in lipase-catalyzed acylation of hydroxyalkylphosphine oxides

The lipase-catalyzed optical resolution of 2-, 3-, and 5-hydroxyalkyl phosphorus compounds 1 provided the corresponding optically pure diastereomers in good yields. (S_P, R)- and (R_P, S)-1 were acylated faster than (S_P, S)- and (R_P, R)-1. The stereoselectivity at the phosphorus atom changed with the flexibility of the active sites in the lipases. The stereoselectivity at the phosphorus atom was higher in the reaction of 1a than in the reaction of 1b,c. The reaction rate of -hydroxyalkylphosphine oxide 1c was faster than that of 1a, although less enantioselectivity was observed at the phosphorus atom.     

Structural determinants of the substrate and stereochemical specificity of phosphotriesterase

Bacterial phosphotriesterase (PTE) catalyzes the hydrolysis of a wide variety of organophosphate nerve agents and insecticides. Previous kinetic studies with a series of enantiomeric organophosphate triesters have shown that the wild type PTE generally prefers the S(P)-enantiomer over the corresponding R(P)-enantiomers by factors ranging from 1 to 90. The three-dimensional crystal structure of PTE with a bound substrate analogue has led to the identification of three hydrophobic binding pockets. To delineate the factors that govern the reactivity and stereoselectivity of PTE, the dimensions of these three subsites have been systematically altered by site-directed mutagenesis of Cys-59, Gly-60, Ser-61, Ile-106, Trp-131, Phe-132, His-254, His-257, Leu-271, Leu-303, Phe-306, Ser-308, Tyr-309, and Met-317. These studies have shown that substitution of Gly-60 with an alanine within the small subsite dramatically decreased k(cat) and k(cat)/K(a) for the R(P)-enantiomers, but had little influence on the kinetic constants for the S(P)-enantiomers of the chiral substrates. As a result, the chiral preference for the S(P)-enantiomers was greatly enhanced. For example, the value of k(cat)/K(a) with the mutant G60A for the S(P)-enantiomer of methyl phenyl p-nitrophenyl phosphate was 13000-fold greater than that for the corresponding R(P)-enantiomer. The mutation of I106, F132, or S308 to an alanine residue, which enlarges the small or leaving group subsites, caused a significant reduction in the enantiomeric preference for the S(P)-enantiomers, due to selective increases in the reaction rates for the R(P)-enantiomers. Enlargement of the large subsite by the construction of an H254A, H257A, L271A, or M317A mutant had a relatively small effect on k(cat)/K(a) for either the R(P)- or S(P)-enantiomers and thus had little effect on the overall stereoselectivity. These studies demonstrate that by modifying specific residues located within the active site of PTE, it is possible to dramatically alter the stereoselectivity and overall reactivity of the native enzyme toward chiral substrates.     

A 5000-Fold Increase in the Specificity of a Bacterial Phosphotriesterase for Malathion through Combinatorial Active Site Mutagenesis

Phosphotriesterases (PTEs) have been isolated from a range of bacterial species, including Agrobcaterium radiobacter (PTE_Ar), and are efficient enzymes with broad substrate ranges. The turnover rate of PTE_Ar for the common organophosphorous insecticide malathion is lower than expected based on its physical properties; principally the pk_a of its leaving group. In this study, we rationalise the turnover rate of PTE_Ar for malathion using computational docking of the substrate into a high resolution crystal structure of the enzyme, suggesting that malathion is too large for the PTE_Ar binding pocket. Protein engineering through combinatorial active site saturation testing (CASTing) was then used to increase the rate of malathion turnover. Variants from a CASTing library in which Ser308 and Tyr309 were mutated yielded variants with increased activity towards malathion. The most active PTE_Ar variant carried Ser308Leu and Tyr309Ala substitutions, which resulted in a ca. 5000-fold increase in k _cat/K _M for malathion. X-ray crystal structures for the PTE_Ar Ser308Leu\Tyr309Ala variant demonstrate that the access to the binding pocket was enhanced by the replacement of the bulky Tyr309 residue with the smaller alanine residue.     

Stereoselective uptake of the GABA-transaminase inhibitors gamma-vinyl gaba and gamma-acetylenic GABA into neurons and astrocytes

The cellular uptake of the GABA-transaminase inhibitors gamma-vinyl GABA (GVG) and gamma-acetylenic GABA (GAG) was studied in cultured neurons and astrocytes. By the use of the individual enantiomersR- andS-GVG andR- andS-GAG it could be shown that in both cell types only theS-enantiomers could be actively transported. Comparing neurons and astrocytes only neurons exhibited a high affinity uptake system forS-GVG (K _m 78.2±20.3 M;V _max 0.71±0.06 nmol · min^–1 · mg^–1 cell protein). In case ofS-GAG it could not be established with certainty whether the neuronal uptake was of the high affinity type. Both GVG and GAG were studied as inhibitors of GABA uptake into neurons and astrocytes.S-GVG andS-GAG were found to be weak inhibitors of GABA uptake suggesting thatS-GVG is not transported by the GABA carrier in neurons. The finding of a much more efficient uptake ofS-GVG into neurons than into astrocytes is in line with the previous observation that neuronal GABA-T is more sensitive than astrocytic GABA-T toS-GVG.     

Role of the second coordination sphere residue tyrosine 179 in substrate affinity and catalytic activity of phenylalanine hydroxylase

Phenylalanine hydroxylase converts phenylalanine to tyrosine utilizing molecular oxygen and tetrahydropterin as a cofactor, and belongs to the aromatic amino acid hydroxylases family. The catalytic domains of these enzymes are structurally similar. According to recent crystallographic studies, residue Tyr179 in Chromobacterium violaceum phenylalanine hydroxylase is located in the active site and its hydroxyl oxygen is 5.1 Å from the iron, where it has been suggested to play a role in positioning the pterin cofactor. To determine the catalytic role of this residue, the point mutants Y179F and Y179A of phenylalanine hydroxylase were prepared and characterized. Both mutants displayed comparable stability and metal binding to the native enzyme, as determined by their melting temperatures in the presence and absence of iron. The catalytic activity (k_cat) of the Y179F and Y179A proteins was lower than wild-type phenylalanine hydroxylase by an order of magnitude, suggesting that the hydroxyl group of Tyr179 plays a role in the rate-determining step in catalysis. The K_M values for different tetrahydropterin cofactors and phenylalanine were decreased by a factor of 3–4 in the Y179F mutant. However, the K_M values for different pterin cofactors were slightly higher in the Y179A mutant than those measured for the wild-type enzyme, and, more significantly, the K_M value for phenylalanine was increased by 10-fold in the Y179A mutant. By the criterion of k_cat/K_Phe, the Y179F and Y179A mutants display 10% and 1%, respectively, of the activity of wild-type phenylalanine hydroxylase. These results are consistent with Tyr179 having a pronounced role in binding phenylalanine but a secondary effect in the formation of the hydroxylating species. In conjunction with recent crystallographic analyses of a ternary complex of phenylalanine hydroxylase, the reported findings establish that Tyr179 is essential in maintaining the catalytic integrity and phenylalanine binding of the enzyme via indirect interactions with the substrate, phenylalanine. A model that accounts for the role of Tyr179 in binding phenylalanine is proposed.Electronic Supplementary Material Supplementary material is available in the online version of this article at Abbreviations AAAHs aromatic amino acid hydroxylases - BH₂ 7,8-dihydro-l-biopterin - BH₄ (6R)-5,6,7,8-tetrahydro-l-biopterin - CD circular dichroism - cPAH Chromobacterium violaceum phenylalanine hydroxylase - DMPH₄ 6,7-dimethyl-5,6,7,8-tetrahydropterin - DTT dithiothreitol - EDTA ethylenediaminetetraacetic acid - ES-MS electrospray ionization mass spectrometry - hPAH human phenylalanine hydroxylase - ICP-AE inductively coupled plasma atomic emission - 6-MPH₄ 6-methyl-5,6,7,8-tetrahydropterin - PAH phenylalanine hydroxylase - PH₄ tetrahydropterin - PKU phenylketonuria - RDS rate-determining step - TH tyrosine hydroxylase - THA 3-(2-thienyl)-l-alanine - TPH tryptophan hydroxylase - wt wild-type     

High-performance liquid chromatographic determination of the enantiomers of cyclophosphamide in serum

A high-performance liquid chromatographic (HPLC) achiral-chiral coupled assay to measure the serum concentration of the enantiomers of cyclophosphamide is described. The R- and S-enantiomers of cyclophosphamide were quantified using a 5-cm-long C₁ Spherisorb 5-μm column, with switching of the eluent containing racemic cyclophosphamide onto a 10-cm-long α₁, acid glycoprotein column. The limit of determination was 1.25 mg l⁻¹ for each enantiomer and the ratio of the enantiomers over the range 2.5 to 100 mg l⁻¹ was 1. Serum enantiomer concentrations in blood samples taken from patients receiving 0.30 to 0.75 g m⁻² of intravenous racemic cyclophosphamide could be measured at least three half-lives post dose. In six patients no significant difference in the clearance of R- and S-cyclophosphamide was found.     

Characterization of (2S,2'R,3'R)-2-(2',3'-[3H]-Dicarboxycyclopropyl)glycine Binding in Rat Brain

Abstract: [(2S,2′R,3′R)-2-(2′,3′-[³H]Dicarboxycyclopropyl)glycine ([³H]DCG IV) binding was characterized in vitro in rat brain cortex homogenates and rat brain sections. In cortex homogenates, the binding was saturable and the saturation isotherm indicated the presence of a single binding site with a K_D value of 180 ± 33 nM and a B_max of 780 ± 70 fmol/mg of protein. The nonspecific binding, measured using 100 µM LY354740, was <30%. NMDA, AMPA, kainate, l (?)-threo-3-hydroxyaspartic acid, and (S)-3,5-dihydroxyphenylglycine were all inactive in [³H]DCG IV binding up to 1 mM. However, several compounds inhibited [³H]DCG IV binding in a concentration-dependent manner with the following rank order of potency: LY341495 = LY354740 > DCG IV = (2S,1′S,2′S)-2-(2-carboxycyclopropyl)glycine > (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid > (2S,1′S,2′S)-2-methyl-2-(2-carboxycyclopropyl)glycine > l -glutamate = ibotenate > quisqualate > (RS)-α-methyl-4-phosphonophenylglycine = l (+)-2-amino-3-phosphonopropionic acid > (S)-α-methyl-4-carboxyphenylglycine > (2S)-α-ethylglutamic acid > l (+)-2-amino-4-phosphonobutyric acid. N-Acetyl-l -aspartyl-l -glutamic acid inhibited the binding in a biphasic manner with an IC₅₀ of 0.2 µM for the high-affinity component. The binding was also affected by GTPγS, reducing agents, and CdCl₂. In parasagittal sections of rat brain, a high density of specific binding was observed in the accessory olfactory bulb, cortical regions (layers 1, 3, and 4 > 2, 5, and 6), caudate putamen, molecular layers of the hippocampus and dentate gyrus, subiculum, presubiculum, retrosplenial cortex, anteroventral thalamic nuclei, and cerebellar granular layer, reflecting its preferential (perhaps not exclusive) affinity for pre- and postsynaptic metabotropic glutamate mGlu2 receptors. Thus, the pharmacology, tissue distribution, and sensitivity to GTPγS show that [³H]DCG IV binding is probably to group II metabotropic glutamate receptors in rat brain.