Identification by high-performance liquid chromatography of tyrosine ammonia-lyase activity in purified fractions of Phaseolus vulgaris phenylalanine ammonia-lyase.

Activities of phenylalanine ammonia-lyase (PAL) and tyrosine ammonia-lyase (TAL) were assessed at each stage of a three-step purification of PAL. Assays were performed by high-performance liquid chromatographic (HPLC) separation and ultraviolet detection of reaction products. Use of HPLC permitted assay of low activities of PAL and TAL for periods up to approximately four and two days, respectively. HPLC also facilitated the accurate quantitation of the product of the TAL reaction, trans-p-coumaric acid, which was observed to isomerize readily under experimental conditions. PAL and TAL were associated throughout the purification procedure, with TAL activity at 0.6-1.3% of PAL activity. It was concluded that, contrary to previous reports, TAL and PAL activities are mediated by the same enzyme, or else by chromatographically very similar enzymes.     

The interaction of heteroaryl-acrylates and alanines with phenylalanine ammonia-lyase from parsley

Acrylic acids and alanines substituted with heteroaryl groups at the beta-position were synthesized and spectroscopically characterized (UV, HRMS, (1)H NMR, and (13)C NMR spectroscopy). The heteroaryl groups were furanyl, thiophenyl, benzofuranyl, and benzothiophenyl and contained the alanyl side chains either at the 2- or 3-positions. While the former are good substrates for phenylalanine ammonia-lyase (PAL), the latter compounds are inhibitors. Exceptions are thiophen-3-yl-alanine, a moderate substrate and furan-3-yl-alanine, which is inert. Possible reasons for these exceptions are discussed. Starting from racemic heteroaryl-2-alanines their D-enantiomers were prepared by using a stereodestructive procedure. From the heteroaryl-2-acrylates, the L-enantiomers of the heteroaryl-2-alanines were prepared at high ammonia concentration. These results can be best explained by a Friedel-Crafts-type electrophilic attack at the aromatic part of the substrates as the initial step of the PAL reaction.     

Gamma irradiation induced enhancement of phenylalanine ammonia-lyase (PAL) and antioxidant activity in peach (Prunus persica Bausch,Cv. Elberta)

Effect of medium dose gamma irradiation on PAL and antioxidant activity of peach fruit was investigated. Peach fruit after harvest at commercial maturity was irradiated in the dose range 1.0–2.0 kGy, stored under refrigerated conditions (3±1 °C, RH 80%) and evaluated at intervals of 7 days. The antioxidant activity as determined by DPPH and FRAP methods revealed significant (p≤0.05) increase particularly in the dose range 1.6–2.0 kGy. During storage, maximum increase in both PAL and antioxidant activity was observed after 21 days. Positive correlation (r=0.75) existed between antioxidant activity and total phenols. EC₅₀ values as obtained from DPPH and FRAP experiments were significantly (p≤0.05) lower in irradiated fruits compared to control.     

Assignment of UVB-responsive cis-element and protoplastization-(dilution-) and elicitor-responsive ones in the promoter region of a carrot phenylalanine ammonia-lyase gene (gDcPAL1)

Expression of a carrot phenylalanine ammonia-lyase (PAL) gene (gDcPAL1) in suspension-cultured carrot cells is induced by dilution of the culture or by application of a fungal elicitor, as well as by ultraviolet B (UVB) irradiation. We demonstrated that among its upstream cis-elements (Takeda et al. [1997] Photochem. Photobiol. 66, 464-470), L4 is UVB responsive, and L1 is protoplastization- (dilution-) and elicitor responsive, from studies with transiently transformed mutated or truncated g-DcPAL1 promoter-luc constructs. This conclusion is consistent with our observation that PAL activities induced by UVB and by protoplastization (dilution) or elicitor are additive.     

Divergent mechanisms of suicide inactivation for ethanolamine ammonia-lyase

Ab initio molecular orbital calculations have been used to study the mechanism of suicide inactivation of ethanolamine ammonia-lyase induced by three different substrate analogues. Analysis of the normal catalytic mechanism with 2-aminoethanol (ethanolamine) as substrate predicts that both the hydrogen-abstraction and hydrogen-reabstraction steps involving the B(12)-cofactor are likely to be exothermic. On the other hand, the proposed inactivation mechanism for the first substrate analogue, glycolaldehyde, leads to a highly stabilized radical that results in a very endothermic (by ca. 90 kJ mol(-)(1)) hydrogen-reabstraction step, which is thought to halt the normal function of the enzyme. Curiously, the energy requirements for a catalytically imposed mechanism in the case of the second substrate analogue, 2-hydroxyethylhydrazine (HEH), parallel those for the catalytic substrate, despite the fact that HEH is found to inactivate EAL experimentally. However, further analysis reveals the presence of a lower energy pathway for HEH that leads to the formation of the highly stabilized hydrazinium radical cation. In a manner similar to when glycolaldehyde is the substrate analogue, this results in an endothermicity for the hydrogen-reabstraction step that is prohibitively large. In contrast to these related inactivation mechanisms, the third substrate analogue, 2-aminoacetaldehyde, apparently accomplishes the inactivation of EAL in an entirely different manner. A pathway for the experimentally observed formation of acetic acid and ammonium cation has been identified and appears catalytic in the sense that 5'-deoxyadenosyl radical is regenerated. However, mechanisms to account for the subsequent formation of 4',5'-anhydroadenosine and degradation of the corrinoid ring of the cofactor have not been elucidated.     

New metallocenic phenylalanine analogs

The syntheses of two new metallocenic analogues of phenylalanine: D,L-β-cymantrenyl alanine and D,L-β-tricarbonyl(cyclobutadienylalanine)iron are described.     

Impact of soil field water capacity on secondary metabolites, phenylalanine ammonia-lyase (PAL), maliondialdehyde (MDA) and photosynthetic responses of Malaysian kacip fatimah (Labisia pumila Benth)

A randomized complete block design 2 × 4 experiment was designed and conducted for 15 weeks to characterize the relationships between production of total phenolics, flavonoid, anthocyanin, leaf gas exchange, total chlorophyll, phenylalanine ammonia-lyase (PAL) and malondialdehyde (MDA) activity in two varieties of Labisia pumila Benth, namely the var. alata and pumila, under four levels of evapotranspiration replacement (ER) (100%; well watered), (75%, moderate water stress), (50%; high water stress) and (25%; severe water stress). The production of total phenolics, flavonoids, anthocyanin, soluble sugar and relative leaf water content was affected by the interaction between varieties and SWC. As the ER levels decreased from 100% to 25%, the production of PAL and MDA activity increased steadily. At the highest (100%) ER L. pumila exhibited signi?cantly higher net photosynthesis, apparent quantum yield, maximum efficiency of photosystem II (f(v)/f(m)) and lower dark respiration rates compared to the other treatment. The production of total phenolics, flavonoids and anthocyanin was also found to be higher under high water stress (50% ER replacement) compared to severe water stress (25% ER). From this study, it was observed that as net photosynthesis, apparent quantum yield and chlorophyll content were downregulated under high water stress the production of total phenolics, flavonoids and anthocyanin were upregulated implying that the imposition of high water stress can enhance the medicinal properties of L. pumila Benth.     

Synthesis of novel benzoxaborole-containing phenylalanine analogues

The synthesis of novel benzoxaborole-containing phenylalanine analogues 2 and 3 has been developed. The key steps involve the preparation of appropriate precursors from the readily available amino acids and the formation of benzoxaborole ring directly in the corresponding amino acid fragment. The resulting compounds 2-3 show improved water solubility at physiological pH, suggesting their potential use as boron delivery agents for boron neutron capture therapy.     

Jet-cooled rotational spectrum of laser-ablated phenylalanine

The rotational spectrum of neutral phenylalanine has been recorded for the first time using laser-ablation molecular-beam Fourier transform microwave spectroscopy (LA-MB-FTMW). Two conformers stabilized by conjugative O-H···N and N-H···π hydrogen bond interactions have been conclusively identified on the basis of experimental values of rotational and (14)N nuclear quadrupole coupling constants. The nonobservation of the rotational spectra of the other low-energy conformers has been attributed to the photofragmentation that takes place in the laser ablation process. Nuclear quadrupole coupling interactions have been used directly to determine the orientation of the amino group and to identify and experimentally characterize the N-H···π interactions.     

Enantioselective synthesis of constrained phenylalanine analogues

Constrained phenylalanine derivatives containing hydrophobic groups and hydrogen bond acceptor and/or donor functionalities were synthesized through a tandem palladium-mediated Heck reaction followed by a rhodium(II)-catalyzed asymmetric hydrogenation. Aryl bromides were found to be better substrates in providing products with higher purity and in good yield. The cesium carbonate-mediated cyclization proceeded smoothly in good yield and optical purity. Aryl iodides reacted selectively over bromides under Jeffery-type conditions (Pd(OAc)₂, Bu₄NCl, Et₃N) providing an opportunity for further metal-mediated functionalization.     

Fluorescence quenching in phenylalanine and model compounds

Abstract— The effects of the carboxylic and amino groups in their neutral and charged forms on the absorption and emission properties of phenylalanine are analyzed with the help of model compounds containing either substituent. Fluorescence yields of phenylalanine and model compounds were measured as a function of pH. Fluorescence quenching occurs in going from COO^- to COOH and from NH₃⁺ to NH₂. Lowering the ionization potential of the phenyl group by substitution increases the quenching effect. This provides more evidence that an intramolecular charge-transfer mechanism may be responsible for intersystem-crossing rate enhancement and fluorescence quenching in phenylalanine and model compounds.     

Tyrosine formation from phenylalanine by ultraviolet irradiation

When phenylalanine was irradiated at ultraviolet (UV) light, p-tyrosine, m-tyrosine and o-tyrosine were identified as hydroxylated products. From p-tyrosine and m-tyrosine, the formation of L-3,4-dihydroxyphenylalanine (DOPA) was observed. The hydroxylation of phenylalanine was prevented by radical scavengers, e.g., catalase, superoxide dismutase, sodium thiocyanate, mannitol, potassium iodide and thiourea. Replacement of air with nitrogen gas prevented the hydroxylation, but did not depress it completely. The addition of H2O2 increased significantly the hydroxylation of phenylalanine. These results suggest that the hydroxylation of phenylalanine by UV irradiation may be caused by .OH formed during the decomposition of H2O.     

Utilization of phenylalanine and phenylacetic acid byPseudomonas solanacearum

Pseudomonas solanacearum utilized phenylalanine as sole source of carbon and nitrogen. Phenylalanine-grown cells rapidly oxidized phenylacetic acid and homogentisic acid. Gentisic and protocatechuic acids were also oxidized, after an initial lag. Phenylacetate-grown cells rapidly oxidized gentisic acid, slowly oxidizedo-hydroxyphenylacetic acid and catechol, and did not oxidizem-hydroxymandelic andm-hydroxyphenylacetic acids. Phenylalanine was degraded via phenylacetic acid-r-lactone andβ ketoadipate; Rothera’s test indicatedortho cleavage of the aromatic ring.     

A new route to fullerene substituted phenylalanine derivatives

A series of fullerene substituted phenylalanine derivatives have been prepared by the condensation of 1,2-(4'-oxocyclohexano)fullerene with ester or Boc protected (4-amino)phenylalanine, H(2)NC(6)H(4)CH(2)CH(COR(1))(NHCOR(2))(where R(1) = OMe, R(2) = Me; R(1) = OH, R(2) = Me, O(t)Bu). Conversion of the imine to the corresponding amine is achieved by di-acid catalyzed hydroboration. Reaction of the N-Ac amino ester with BBr(3) led to the formation of the parent amino acid, while the Boc-protected derivative readily undergoes coupling with NH(2)-Gly-OEt. The reduction of the imine is not accompanied by hydroboration of the fullerene cage.     

CdTe纳米晶与苯丙氨酸相互作用的研究

以巯基乙酸为稳定剂,在水相中合成了CdTe纳米晶,通过考察红外光谱、紫外光谱和光致发光光谱的变化,确定了苯丙氨酸(Phe)对CdTe纳米晶的光致发光效率以及稳定性的影响.结果表明苯丙氨酸与CdTe纳米晶之间存在着较强的相互作用,这种作用导致CdTe纳米晶的发光强度增大,稳定性增强,苯丙氨酸不仅是配位剂,它同时又起到了稳定剂的作用,并初步确认这种加强和稳定作用来自于苯丙氨酸和CdTe纳米晶之间所形成的配位键和静电作用以及苯丙氨酸与巯基乙酸之间形成的氢键.此外,苯丙氨酸的特殊结构对防止纳米晶的聚集有很大作用.