Phenylalanine ammonia-lyase: Mirror-image packing of d- and l-phenylalanine and d- and l-transition state analogs into the active site

The binding of substrate and product analogs to phenylalanine ammonia-lyase (EC 4.3.1.5) from maize has been studied by a protection method. The ligand dissociation constants, K_L, were estimated from the variation with [L] of the pseudo-first-order rate constants for enzyme inactivation by nitromethane. The phenylalanine analogs d- and l-2-aminooxy-3-phenylpropionic acid showed K_L, values over 20,000-fold lower than the K_m for l-phenylalanine. From these and other K_L values it is deduced that when the enzyme binds l-phenylalanine the structural free energy stored in the protein is higher than when it binds the superinhibitors. Models for binding d- and l-phenylalanine and the superinhibitors are described. The enantiomeric pairs are considered to have similar K_L values because they pack into the active site in a mirror-image relationship. If the elimination reaction approximates to the least-motion course deduced on stereoelectronic grounds, the mirror-image packing of the superinhibitors into the active site mimics the conformation inferred for a transition state in the elimination. It appears, therefore, that structural changes take place in the enzyme as the transition state conformation is approached causing stored free energy to be released. This lowers the activation free energy for the elimination reaction and accounts for the strong binding by the above analogs.     

l-DOPA (l-3,4-dihydroxyphenylalanine) uptake by human red blood cells

The uptake of l-DOPA (l-3,4-dihydroxyphenylalanine) was studied in normal human red blood cells in vitro using l-[3-¹⁴C]DOPA. Uptake was slow, tending towards a distribution ratio close to unity with a half-time to equilibrium of one hour. Uptake was not Na⁺-dependent. Concentration dependence studies showed both saturable and non-saturable components of uptake, and inhibition studies using l-leucine and l-tryptophan suggest that the L and T systems of red cell amino acid uptake are involved. A powerful inhibitor of both systems, 3,4-dihydroxy-2-methylpropriophenone (U-0521), is described. It is concluded that uptake is by carrier-mediated facilitated diffusion via the L and T systems for which l-DOPA has low affinity.     

Structure of l-arabino-d-galactan-containing glycoproteins from radish leaves

Two l-arabino-d-galactan-containing glycoproteins having a potent inhibitory activity against eel anti-H agglutinin were isolated from the hot saline extracts of mature radish leaves and characterized to have a similar monosaccharide composition that consists of l-arabinose, d-galactose, l-fucose, 4-O-methyl-d-glucuronic acid, and d-glucuronic acid residues. The chemical structure features of the carbohydrate components were investigated by carboxyl group reduction, methylation, periodate oxidation, partial acid hydrolysis, and digestion with exo- and endo-glycosidases, which indicated a backbone chain of (1→3)-linked β-d-galactosyl residues, to which side chains consisting of α-(1→6)-linked d-galactosyl residues were attached. The α-l-arabinofuranosyl residues were attached as single nonreducing groups and as O-2- or O-3-linked residues to O-3 of the β-d-galactosyl residues of the side chains. Single α-l-fucopyranosyl end groups were linked to O-2 of the l-arabinofuranosyl residues, and the 4-O-methyl-β-d-glucopyranosyluronic acid end groups were linked to d-galactosyl residues. The O-α-l-fucopyranosyl-(1→2)-α-l-arabinofuranosyl end-groups were shown to be responsible for the serological, H-like activity of the l-arabino-d-galactan glycoproteins. Reductive alkaline degradation of the glycoconjugates showed that a large proportion of the polysaccharide chains is conjugated with the polypeptide backbone through a 3-O-d-galactosylserine linkage.     

Silk fibroin model,poly(l-alanylglycyl-l-alanylglycyl-l-alanylglycyl-l-seryglycine)

l-Alanylglycyl-l-alanylglycyl-l-alanylglycyl-l-serylglycine and its pentachlorophenyl ester methanesulphonate have been synthesized as monomers for the preparation of silk fibroin model polypeptide. The former octapeptide was polymerized with diphenylphosphorylazide (DPPA) and triethylamine in DMSO or in HMPA—pyridine, and the latter octapeptide pentachlorophenylester was polymerized by adding triethylamine in DMSO to give poly(l-alanylglycyl-l-alanylglycyl-l-alanylglycyl-l-serylglycine). This sequential polypeptide gave a similar i.r. pattern to the crystalline part of Bombyx mori silk fibroin, which indicated antiparallel β-conformation. Dialysis of the solution of this polymer in 60%, aqueous LiBr against water gave mainly the polymer of α-form. O.r.d. measurements suggest that this polypeptide exists as a random structure in dichloroacetic acid on in 60% aqueous LiBr.     

Morphology and structure of γ-benzyl-l-glutamate copolymerized with l-phenylalanine, l-valine and l-alanine

Observation of random copolypeptides of γ-benzyl-l-glutamate with l-phenylalanine, l-valine and l-alanine was carried out in an electron microscope with samples cast from dilute solution. The relationship between the morphology and the molecular conformation in solution was studied with mixed solvents composed of chloroform and trifluoroacetic acid; these show a preference for α-helix and random coil, respectively. From the solutions in which molecules take α-helical conformation, fibrous films of nematic structure were formed. From random coil solutions discrete precipitates with folded molecules such as lamellar single crystals, piles of lamellae and structureless particles were formed. A copolypeptide containing l-valine in sufficiently large quantity to form β-structure also showed a variation in morphology with solvent, from films to discrete precipitates. It is suggested that the change in stiffness of the molecules contributes to the morphological variation.     

Differential fixation of poly(L-arginine) and poly(L-lysine) by tannic acid and its application to the fixation of collagen in electron microscopy

A differential fixation of poly(L-arginine) and poly(L-lysine) has been demonstrated by means of cellulose acetate electrophoresis and colorimetric titration. Electrophoresis showed that at pH 3.0 and concentrations between 0.025% and 2% the reagent interacts with poly(L-arginine) but not with poly(L-lysine). at pH 7.5, however, poly(L-lysine) also reacts, although at a higher concentration of tannic acid than was required to fix poly(L-arginine) at this pH. Colorimetric titration revealed that for poly(L-arginine) the reaction with tannic acid commences at pH 3.0 and is complete at pH 4.1 whereas for poly(L-lysine) the reaction commences at pH 3.5 and is complete at pH 4.9. It is suggested that the reaction is predominantly electrostatic. The results are discussed in relation to the use of tannic acid as a protein fixative in electron microscopy.     

Separate binding sites in rat brain synaptic membranes for l-cysteine sulfinate and for l-glutamate

Binding of l-[³H]cysteine sulfinic acid (CSA) and l-[³H]glutamate were compared in various subcellular fractions and in the presence of a variety of pharmacological and ionic manipulations in order to test the possibility that the two amino acids possessed separate binding sites.The specific l-[³H]cysteine sulfinate binding was found to be enriched maximally in medium and high density synaptic membranes, while the crude mitochondrial synaptosomal fraction displayed the highest l-[³H]glutamate binding. The ratio of l-[³H]cysteine sulfinate binding/l-[³H]glutamate binding was variable across brain regions. Several compounds differentially affected l-[³H]cysteine sulfinate and l-[³H]glutamate binding. l-cysteine sulfinate was the most potent displacer regardless of the binding considered. Finally, while cations produced qualitatively similar effects on the binding of the two amino acids, quantitative differences were evident.In sum, these data revealed the complexity of l-[³H]cysteine sulfinate and l-[³H]glutamate binding. They suggest the existence of several binding sites and that some of these are shared by both substances. However, the results also indicate that separate binding sites for the two amino acids exist in synaptic membrane, giving further support to the hypothesis that cysteine sulfinate serves a neurotransmitter role in the central nervous system.     

Selenium derivatives of L-arabinose, D-ribose,and D-xylose

Attempted cyclization of 2,3,4-tri-O-methyl-5-seleno-L-arabinose dimethyl acetal in acidic solution gave the corresponding diselenide. Intramolecular attack by the selenobenzyl group at C-5 of 5-O-p-tolylsulfonyl-L-arabinose dibenzyl diseleno-acetal resulted in the formation of benzyl 1,5-diseleno-L-arabinopyranoside. Similarly, 2,3,5-tri-O-methyl-4-O-p-tolylsulfonyl-D-xylose dibenzyl diselenoacetal gave benzyl 2,3,5-tri-O-methyl-1,4-diseleno-L-arabinofuranoside, and 2,3,4-tri-O-acetyl-5-O-p-tolylsulfonyl-D-xylose (or ribose) dibenzyl diselenoacetal gave benzyl 2,3,4-tri-O-acetyl-1,5-diseleno-D-xylo- (or ribo-)pyranoside. The glycosylic benzylseleno group was removed from the pyranoside with mercuric acetate, but attempted deacetylation of the product led to decomposition and not to the expected 5-seleno-D-xylopyranose.     

The efflux of l-carnitine from cells in culture (CCL 27)

The efflux of l-[³H]carnitine was studied in cells from an established cell line from human heart (Girardi human heart cells, CCL 27). The cells were loaded with 4 μmol/l l-[³H]carnitine for 1 or 24 h, and the efflux of radioactivity into the medium was measured. The amount of intracellular l-[³H]carnitine retained was expressed as a function of time. The results were fitted to an exponential equation, from which efflux rate constants were computed.Increasing the extracellular concentration of butyrobetaine, l-carnitine, d-carnitine, betaine, dl-norcarnitine or 3-dimethylamino-2-hydroxypropionic acid each increased the observed efflux. This is most likely due to accelerated exchange diffusion. The substrate specificity of this accelerated exchange diffusion is different from what previously has been found in competitive uptake studies of l-carnitine. l-Carnitine was preferentially released to l-acetylcarnitine, and blocking the sulfhydryl groups with 5,5-dithiobis(2-nitrobenzoic acid) increased the efflux.     

Kinetics and mechanism of oxidation of d-fructose and l-sorbose by chromium(VI) and vanadium(V) in perchloric acid medium

Kinetic data for the oxidations of d-fructose and l-sorbose by chromium(VI) and vanadium(V) in perchloric acid medium are reported. The addition of perchloric acid and sodium perchlorate increases the pseudo-first-order rate constants. Change of the reaction medium from water to deuterium oxide appreciably affects the rates of chromium(VI) oxidations, but does not affect those of vanadium(V) oxidations. The activation parameters are ΔH3 = 46.6 ±3.4 (fructose) and 50.6 ±6.3 (sorbose) kJ.mol^?1, and ΔS3 = ?105 ±11 (fructose) and ?100 ±20 (sorbose) J.deg^?1.mol^?1 for chromium(VI) oxidations, and, for the other reactions, ΔH3 = 53.2 ±4.2 (fructose) and 52.3 ±6.3 (sorbose) kJ.mol^?1, and ΔS3 = ?139.0 ±14 (fructose) and ?137 ±20 (sorbose) J.deg^?1.mol^?1. The kinetics of the oxidations of ketohexoses by chromium(VI) indicate no intermediate-complex formation, whereas those for vanadium(V) indicate the formation of a 1:1 intermediate complex between ketohexoses and vanadium(V).     

Alkali and oxygen-alkali treatment of D-arabino-hexosulose and O-β-D-glucopyranosyl-(1 → 4)-d-arabino-hexosulose.

Benzilic acid rearrangement of D-arabino-hexosulose (1) and O-β-D-glucopyranosyl-(1→4)-D-arabino-hexosulose (2) favours formation of mannonic acid and mannonic acid moieties, respectively. The results show that formation of aldonic acid end-groups via terminal aldosulose moieties is of little importance during oxygen-hydrogencarbonate treatment of (1→4)-linked polysaccharides. The major reaction of 1 in the absence of oxygen involves loss of C-1 as formic acid. The enediol intermediate gives rise to pentoses and pentuloses (degraded completely at high alkalinity), and 3-deoxypentonic acids. The yield of 3-deoxypentonic acids is decreased in the presence of oxygen, whereas that of arabinonic, erythronic, and glycolic acids is increased. The main reaction of 2 giving rise to aliphatic hydroxy acids is β-elimination of the glucose moiety, yielding a tricarbonyl intermediate (3) which, in sodium hydrogencarbonate, is decomposed mainly to 3,4-dihydroxybutanoic and glycolic acids. In sodium hydroxide, 3-deoxypentonic acids are among the major reaction products. In addition, a complex mixture of u.v.-absorbing solutes is formed, some of which are held irreversibly by anion exchangers.     

Solvolysis of charged active esters of carboxylic acids with cyclo (l- or d-Glu-l-His)

Cyclic dipeptide cyclo(l- or d-Glu-l-His) carrying an anionic site and a nucleophilic site has been synthesized and used as a catalyst for the solvolysis of cationic esters in aqueous alcohols. In the solvolysis of 3-acyloxy-N-trimethylanilinium iodide (S⁺_n, n = 2 and 10) and Cl^?H₃N⁺(CH₂)₁₁COOPh(NO₂), no efficient nucleophilic catalysis was observed. On the other hand, in the solvolysis of Gly-OPh(NO₂)·HCl, Val-OPh(NO₂)·HCl and Leu-OPh(NO₂)·HCl a very efficient general base-type catalysis by cyclo(l-Glu-l-His) was observed. In particular, with the latter two substrates the catalysis by cyclo(l-Glul-His) was more efficient than that by imidazole, although the catalysis was not enantiomer-selective. The diastereomeric cyclic dipeptide cyclo(d-Glu-l-His) was almost inactive under the same conditions. Confomation of cyclo(l- or d-Glu-l-His) in aqueous solution was investigated and the structure/catalysis relationship is discussed.     

Synthesis and conformational analysis of cyclo-TRI[l-valyl-d-hexahydromandelyl]

The synthesis of the cyclo-hexadepsipeptide [l-valyl-d-hexahydromandelyl]₃ is described. Examination of this macrocyclic compound by 220-MHz nuclear magnetic resonance spectroscopy shows that symmetrical conformations are stabilized in strongly polar solvents (trifluoroacetic acid, acetonitrile), whereas asymmetric conformations are preferred in nonpolar or slightly polar media such as carbon tetrachloride, chloroform, cyclohexane, and benzene.From analysis of the temperature dependence of the chemical shift and of the coupling constants, together with conformational energy calculations, a model is proposed for the preferred conformation of this molecule in nonpolar solvents.     

Glycans from streptococcal cell-walls: the molecular structure of an antigenic diheteroglycan of D-glucose and L-rhamnose from Streptococcus bovis

The monosaccharide sequence and glycosidic bond-types have been determined for an antigenic diheteroglycan of D-glucose and L-rhamnose from the cell wall of Streptococcus bovis, strain C3, by use of an integrated analytical scheme based on methylation analysis, periodate oxidation, oxidation with chromium trioxide, enzymic hydrolysis, and chemical degradation. A typical molecule of the glycan consists of a main chain of L-rhamnosyl residues and isomaltose side-chains, with 16 repetitions of the structure, -α-L-rhamnosyl-(1→3)-[α-D)-glucosyl-(1→6)-α-D-glucosyl-(1→2)]-α-L-rhamnosyl-(1→2)-α-L-rhamnosyl-, linked alternately by α-L-(1→3) and α-L-(1→2) linkages. The isomaltose side-chains of the glycan are the immunodeterminant groups. The new antigenic glycan is ideally suited for use in the preparation of anti-isomaltose antibodies, which should be of value in the detection of other antigens having isomaltose determinants.     

Microdetermination of d-amino acids and d-amino acid oxidase activity with 3-methyl-2-benzothiazolone hydrazone hydrochloride

A simple and rapid technique for the determination of the d-amino acids which are oxidized by d-amino acid oxidase has been presented. This method involves an oxidation of d-amino acids with d-amino acid oxidase in the presence of catalase, and the spectrophotometric determination of the resultant α-keto acids with MBTH. The additions of l-amino acids have no influence on the quantitative estimation of d-amino acids. The method is suitable for the assay of d-amino acids in the presence of the l isomers, and is also applicable for the determination of d-amino acid oxidase activity.     

Convenient,laboratory procedure for producing solid d-arabino-hexos-2-ulose (d-glucosone)

The production of solid d-arabino-hexos-2-ulose (d-glucosone) from d-glucose by use of an enzyme, pyranose-2-oxidase (EC 1.1.3.10), is described. The enzyme is extracted from the mycelia of Polyporus obtusus, partially purified, and then immobilized on activated CH-Sepharose 4B. The enzymic conversion of d-glucose into d-glucosone is simple and convenient, and provides a product free from residual d-glucose. Lyophilization of the filtered reaction-solution yields the product, solid d-glucosone. Assay methods have been developed for monitoring the enzymic reaction and evaluating the purity of the final product.     

The effect of areneboronic acids on the alkaline conversion of d-glucose into d-fructose

Benzeneboronic acid, 4-methoxybenzeneboronic acid, 3-nitrobenzeneboronic acid, and sulphonated benzeneboronic acid have been used to displace the pseudo-equilibria established in aqueous alkali between d-glucose, d-fructose, and d-mannose to give greatly increased yields of d-fructose. The effect of reaction temperature, pH, overall concentration, and molar ratio of acid:sugar on the yield of d-fructose has been investigated by using an automated assay for d-fructose.     

Non-pyridine nucleotide dependent l-(+)-glutamate oxidoreductase in Azotobacter vinelandii

l-(+)-Glutamate oxidation that is non-pyridine nucleotide dependent is readily carried out by a membrane-bound enzyme in Azotobacter vinelandii strain O. Enzyme activity concentrates in a membranous fraction that is associated with the Azotobacter electron transport system. This l-glutamate oxidation is not dependent on externally added NAD⁺, NADP⁺, FAD, or FMN for activity. O₂, phenazine methosulfate and ferricyanide all served as relatively good electron acceptors for this reaction; while cytochrome c and nitrotetrazolium blue function poorly in this capacity. Paper chromatographic analyses revealed that the 2,4-dinitrophenylhydrazine derivative formed from the enzymatic oxidation of l-glutamate was α-ketoglutarate, while microdiffusion studies indicated that ammonia was also a key end product. These findings suggest that the overall reaction is an oxidative deamination. Ammonia formation was found to be stoichiometric with the amount of oxygen consumed (2 : 1 respectively, on a molar basis). The oxidation of glutamate was limited to the l-(+)-enantiomer indicating that this reaction is not the generalized type carried out by the l-amino acid oxidase. This oxidoreductase is functionally related to the Azotobacter electron transport system: (a) the activity concentrates almost exclusively in the electron transport fraction; (b) the l-glutamate oxidase activity is markedly sensitive to electron transport inhibitors, i.e. 2-n-heptyl-4-hydroxyquinoline-N-oxide, cyanide, and 4,4,4-trifluoro-1-(2-thienyl)-1,3-butanedione; and (c) spectral studies on the Azotobacter R₃ fraction revealed that a substantial amount of the flavoprotein (non-heme iron) and cytochrome (a₂, a₁, b₁, c₄ and c₅) are reduced by the addition of l-glutamate.     

Structure of the D-fructan isolated from garlic (Allium sativum) bulbs

Extraction of defatted garlic bulbs with hot water yielded a mixture of polysaccharides containing pectic acid, a D-galactan, and a fructan component. The pectic acid was partially removed as calcium pectate, and the galactan-enriched portion was separated by fractional precipitation with alcohol; on concentration and several fractionations, the supernatant liquor furnished the fructan component, which contained fructose (94.4%) and glucose (4.3 %). Methanolysis and hydrolysis of the permethylated fructan gave (a) 1,3,4,6-tetra-O-methyl-D-fructose, (b) 2,3,4,6-tetra-O-methyl-D-glucose, (c) 2,4,6-tri-O-methyl-D-glucose, and (d) 3.4,6-tri-O-methyl-D-fructose in the ratio (a + b): (d) = 1:20.3. On periodate oxidation, the fructan reduced one molar equivalent of the oxidant per hexosyl residue, and liberated one molar equivalent of formic acid per 51 hexosyl residues. On Smith degradation, the major product was glycerol, and ～2 % of the glucose survived. From these results, and from the fact that the fructan is hydrolyzed by β-D-fructofuranosidase, a linear, inulin-type of structure is suggested for it.     

Conformational studies on synthetic polypeptides: poly (d-phenylglycine) and poly(d-cyclohexylglycine)

Poly(d-phenylglycine) and poly(d-cyclohexylglycine) containing phenyl and cyclohexyl rings bound to the α-carbon of the polypeptide chain, have been synthesized. Circular dichroism measurements show that both polymers undergo a conformational transition from the random-coil form to an ordered form, upon addition of water, ethanol or trifluoroethanol to sulphuric acid solutions. Solid state measurements indicate that the ordered structures of poly(d-phenylglycine) and poly(d-cyclohexylglycine) are of the β-type. While for the former the antiparallel arrangement is predominant, for the latter there seems to be a greater tendency towards the parallel form. The ordered form of poly(d-cyclohexylglycine) is slightly more stable than the corresponding form of poly(d-phenylglycine) in all the above solvent systems. This can be interpreted in terms of stronger non covalent bond formation in the former polypeptide. Our results have been compared with literature on poly(l-phenylalanine) and poly(l-cyclohexylalanine).