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.     

X-ray diffraction study of the molecular association in aqueous solutions of d-fructose, d-glucose,and sucrose

X-ray diffractograms of aqueous solutions of d-fructose, d-glucose, and sucrose, and of two amorphous “solid” forms of these carbohydrates are recorded in the angular range of O 2-16°. The formation, in these diffractograms, of one or two intensity maxima as the concentration is varied is interpreted in terms of modification of the molecular association. A model is proposed that takes account of the state of organization of the solutions of the three sugars as a function of the concentration. Sucrose shows an “anomaly”, as it is the only sugar to establish intramolecular bonds. The number of these intramolecular bonds also depends on the concentration.     

Synthesis and growth regulator activity of fatty acyl derivatives of d-glucose and d-galactose

In an attempt to gain information about one or more components of the brassin complex, fatty acid esters of d-glucose and d-galactose were prepared and tested for growth regulator activity in a bean hypocotyl bioassay. 4-O-Acyl-d-glucoses and, perhaps, 1-O-acyl- d-galactoses had a similar qualitative activity to that of the brassin complex. 3-O-Acyl- d-galactoses inhibited elongation of bean hypocotyls and stimulated rooting. 3- And 6-O- acyl-d-glucoses both stimulated and inhibited elongation, depending on the source of fatty acids; in both cases, stimulation was observed when safflower oil was used as the source of fatty acids and inhibition was observed when peanut oil was used as the source of fatty acids. Fatty alkyl β-d-galactopyranosides were inactive.     

2,5-anhydro-d-hexitols: syntheses of 2,5-anhydro-d-altritol and 2,5-anhydro-d-iditol

2,5-Anhydro-d-altritol (2a) and the previously-unknown 2,5-anhydro-d-iditol (3a) have been prepared from 2,5-anhydro-d-mannitol (1a). The preparation of 3a from the intermediate epoxide 7b is particularly sensitive to pH, and a mechanism is proposed to explain this. Attention is drawn to the limitations of the trifluoroperacetic acid-disodium hydrogenphosphate procedure for the epoxidation of alkenes of diminished reactivity.     

An aminotransferase specific for the d-enantiomorph of methionine

An enzyme was isolated from germinating peanut seed and shown to be an aminotransferase specific for the d-enantiomorph of methionine. The keto acid of methionine, α-keto-γ-methylthiobutyrate, was isolated from the reaction mixture and identified. Of the keto acids tested pyruvic acid was the most effective acceptor for the amino group of methionine. A small amount of enzyme was isolated which gave only one band on disc gel electrophoresis.     

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.     

Phenylalanine ammonia-lyase: A model for the cooperativity kinetics induced by d- and l-phenylalanine

A preparation of l-phenylalanine ammonia-lyase (EC 4.1.3.5.) from soybean (Glycine max L. cv. Kanrich) showed negative cooperativity with respect to l-phenylalanine and competitive inhibition by d-phenylalanine. A two-protomer partially concerted model for inhibition kinetics is described. If cooperativity is associated with ligand binding but not k_cat, plots of v against log [S] at constant [I] are symmetrical. Such curves may be fitted by graphical or iterative least-squares methods. The experimental results conform to this restricted model. The three-substrate and three-inhibitor dissociation constants were estimated by a stepwise procedure. For substrate only the first and second dissociation constants were 12 and 78 μm, respectively, with a symmetry point value of 30.5 μm. To a first approximation, site occupancy determines the cooperativity. As d- and l-phenylalanine produce equivalent effects, they are assumed to pack into the same induced space. As ligand binding at one site has little influence on the relative d:l binding at the other and does not influence k_cat, cooperativity probably reflects changes in regions remote from the active site such as the interface between the protomers. The regulatory range in [S] of the enzyme in vivo may be indicated by the linearity range of the semilog plot for the isolated enzyme. The observed range corresponds to a 100-fold change in [S] compared to a 10-fold change for Michaelis-Menten kinetics.     

A microplate assay for d-xylose/ d-glucose isomerase

A modification of the resorcinol method of Kulka¹ for the determination of ketoses is described. Though being a stopped enzyme test, it is much more sensitive than the carbazol method and by applying microtiter plates and measuring with an ELISA reader, a large number of tests can be performed within a short time, thereby facilitating initial velocity studies.

The test is linear up to a concentration of 2.5 m -xylulose even in the presence of 10 m -xylose and 2 m -fructose in the presence of 10 m -glucose. The sensitivity is 25 μ for xylulose and 38 μ for fructose. The test method is insensitive to perturbations of substances frequently used in isolation procedures such as ammonium sulfate, Triton X-100, PEG 6000, sodium dodecyl sulfate, and ethanol in moderate concentrations.     

A sensitive,specific radioisotope assay for l-glutamine-d-fructose-6-phosphate aminotransferase

A sensitive and specific radioassay for l-glutamine-d-fructose-6-phosphate aminotransferase (EC 5.3.1.19) activity is presented. Picomoles of product are measurable, and the assay can be applied to systems having limited quantities of available protein, particularly in extracts of either cell or organ cultures. The assay is at least 10,000 times more sensitive under K₁ concentrations of fructose 6-phosphate than the modified Elson-Morgan colorimetric assay and 20 times more sensitive under saturating conditions of fructose 6-phosphate. As little as 0.5 μg of cell-extract protein will yield measurable product. In contrast, 280 μg of crudeextract protein from colon is required with the modified Elson-Morgan colorimetric assay.     

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.     

Kinetics of base-catalyzed degradation of phenyl d-gluco-pyranosides

The kinetics of base-catalyzed cleavage of the glycosidic linkage in phenyl α- and β-d-glucopyranoside have been studied at various concentrations of base, and are interpreted in terms of formation of anionic species as reactive intermediates, accompanied by a minor mechanism of bimolecular, nucleophilic substitution.The data indicate that, in <M sodium hydroxide solution, more than 80% of the cleavage reaction proceeds through an intramolecular-displacement process facilitated by the anchimeric assistance of the hydroxyl group at C-6, and C-2, for the α- and β-d-glycoside, respectively. However, the bimolecular substitution reaction becomes increasingly apparent as the alkalinity is raised above 1.5M concentration of base.     

Laser-raman spectra of d-fructose in aqueous solution

Laser-Raman spectra of d-fructose in water at different concentrations were recorded, and assignments of the frequencies were proposed, based on earlier work on the Raman spectra of other sugars, and determination by other techniques of the composition of aqueous solutions of d-fructose as regards different isomers. It was found that the frequencies of vibration of the furanoid are higher than those of the pyranoid ring. The proportions of the furanoses and pyranoses, found from the ratio of the Raman intensities for the same modes of vibration, were similar to those found by other techniques. Shifts of intensities and frequencies were observed in the region of OH and CH bands, and were assigned to probable association between molecules of d-fructose and water.     

Facile synthesis of d-lividosamine

A simple, four-step synthesis of d-lividosamine starting from N-acetyl-d-glucosamine via a furanosyl oxazoline intermediate is described.     

Synthesis of acetylenic sugars and uronic acids via two-carbon chain extension of d-ribose

Treatment of methyl β-d-ribofuranoside with acetone gave methyl 2,3-O-isopropylidene-β-d-ribofuranoside (1, 90%), whereas methyl α-d-ribofuranoside gave a mixture (30%) of 1 and methyl 2,3-O-isopropylidene-α-d-ribofuranoside (1a). On oxidation, 1 gave methyl 2,3-O-isopropylidene-β-d-ribo-pentodialdo-1,4-furanoside (2), whereas no similar product was obtained on oxidation of 1a. Ethynylmagnesium bromide reacted with 2 in dry tetrahydrofuran to give a 1:1 mixture (95%) of methyl 6,7-dideoxy-2,3-O-isopropylidene-β-d-allo- (3) and -α-l-talo-hept-6-ynofuranoside (4). Ozonolysis of 3 and 4 in dichloromethane gave the corresponding d-allo- and l-talo-uronic acids, characterized as their methyl esters (5 and 6) and 5-O-formyl methyl esters (5a and 6a). Ozonolysis in methanol gave a mixture of the free uronic acid and the methyl ester, and only a small proportion of the 5-O-formyl methyl ester. Malonic acid reacted with 2 to give methyl 5,6-dideoxy-2,3-O-isopropylidene-β-d-ribo-trans-hept-5-enofuranosiduronic acid (7).     

A facile synthesis of 5-thio-l-fucose and 5-thio-d-arabinose from d-arabinose

5-Thio-l-fucopyranose tetraacetate was synthesized in 11 steps from or d-arabinose diethyl dithioacetal by one-carbon elongation at C-5. Highly diastereo-selective addition of MeLi in ether to a derivative was achieved to give the corresponding 6-deoxy-β-d-altrofuranose isomer in good yield. A sulfur atom was introduced at C-5 of 6-deoxy-d-altrofuranose derivatives via substitution of a 5-tosylate with KSAc in HMPA with inversion of configuration, giving 5-thio-l-fucopyranose. A derivative was also prepared from 6-deoxy-β-d-altrofuranose derivatives. 5-Thio-d-arabinopyranose tetraacetate, the 5-demethyl analog of 5-thio-l-fucose, was also synthesized from in 5 steps. 5-Thio-d-arabinose showed weak inhibitory activity against α-l-fucosidase from bovine kidney (K_{i = 0.77 mM}).     

Formation of 3-octuloses by a self-aldol reaction of d-erythrose

When kept at 105° for 2.5 h, weakly alkaline, syrupy d-erythrose was readily converted into a mixture containing mainly d-glycero-tetrulose, the previously unknown β-d-altro-l-glycero-3-octulofuranose (2), and α-d-gluco-l-glycero-3-octulopyranose, which were isolated as the corresponding acetates. Treatment of 2 with Dowex 50 (H⁺) resin yielded 3,8-anhydro-β-d-altro-l-glycero-octulopyranose, identified as its acetate. Previous discrepancies in the [α]_d values for d-erythrose appear partly to originate in the self-aldol reaction. The dimerisation of d-erythrose 4-phosphate is also described.     

d-Glucose- and d-mannose-based antimetabolites. Part 2. Facile synthesis of 2-deoxy-2-halo-d-glucoses and -d-mannoses

Modified d-glucose and d-mannose analogs are potentially clinically useful metabolic inhibitors. Biological evaluation of 2-deoxy-2-halo analogs has been impaired by limited availability and lack of efficient methods for their preparation. We have developed practical synthetic approaches to 2-deoxy-2-fluoro-, 2-chloro-2-deoxy-, 2-bromo-2-deoxy-, and 2-deoxy-2-iodo derivatives of d-glucose and d-mannose that exploit electrophilic addition reactions to a commercially available 3,4,6-tri-O-acetyl-d-glucal.     

d-Glucose- and d-mannose-based antimetabolites. Part 2. Facile synthesis of 2-deoxy-2-halo-d-glucoses and -d-mannoses

Modified d-glucose and d-mannose analogs are potentially clinically useful metabolic inhibitors. Biological evaluation of 2-deoxy-2-halo analogs has been impaired by limited availability and lack of efficient methods for their preparation. We have developed practical synthetic approaches to 2-deoxy-2-fluoro-, 2-chloro-2-deoxy-, 2-bromo-2-deoxy-, and 2-deoxy-2-iodo derivatives of d-glucose and d-mannose that exploit electrophilic addition reactions to a commercially available 3,4,6-tri-O-acetyl-d-glucal.     

A facile synthesis of d-ribo-C20-phytosphingosine and its C2 epimer from d-ribose

A facile synthetic route to d-ribo-C₂₀-phytosphingosine 31 and its C2 epimer 32 is described. The Overman rearrangement of allylic trichloroacetimidates derived from the known ribose derivative 7 has been used as the key step. The subsequent functional group interconversions in rearranged products 14 and 15 followed by Wittig olefination, Pd/C-mediated reduction and the removal of protecting groups successfully constructed the final molecules.     

Isomerization of d-glucose with sodium aluminate: Mechanism of the reaction

A mechanism for the isomerization of d-glucose to d-fructose by sodium aluminate is proposed, involving transformation of a β-d-glucopyranose-1,3-aluminate complex into an α-d-fructofuranose-1,3,6-aluminate complex through an enolaluminate complex that inhibits the formation of a d-mannose-aluminate complex. The α-d-fructofuranose-1,3,6-aluminate further reacts to form a d-psicose-aluminate complex in substantial yield. Constant degradation of the 6-carbon sugars occurred during the reaction because of the high pH of the solution. The C₆ sugars were analyzed chromatographically but the degradation products were not identified.