4-Pyridoxylic acid

The high toxicity of AOA is due to its very high efficiency as a transaminase inhibitor (K =0.45 pM) as compared to its efficacy as a PAL inhibitor (K. = 120 pM) (48), making it impossible to effectively inhibit PAL iti vivo without also greatly inhibiting amino acid metabolism. Other pyridoxyl phosphate-requiring enzymes, such as ACC synthase (an enzyme involved in ethylene production) (49), are also more sensitive to AOA than to AOPP. 

Amino acids undergo a condensation reaction with pyridoxal in alkaline medium to form a Schiff base which can be converted into stable pyridoxyl-amino acids by catalytic reduction or by reduction with sodium tetrahydroborate. The reactions involved are illustrated in Fig. 4.46. The resulting derivatives can be detected in quantities as low as 5-10"10 moles by fluorescence at 332 nm (excitation) and 400 nm (emission). Column chromatography may be used to separate die pyridoxyl-amino acid derivatives [93,94]. 

Method. Solutions of amino acids in phosphate buffer (pH 9.3) are mixed with an equal volume of freshly prepared 0.4 M pyridoxal solution (adjusted to pH 9.3) and permitted to stand at 8 °C for 30 min. (The molar ratio of pyridoxal to amino acid should be >75 1.) At this point, 1 ml of sodium tetrahydroborate solution (100 mg/ml in 0.1 N sodium hydroxide) is added and the contents are gently shaken. Excess of sodium tetrahydroborate is destroyed by addition of sufficient hydrochloric acid (pH 1-2) prior to column chromatography. The pyridoxal derivatives are separated on a column (100 X 0.6 cm) of Aminex A-5 ion-exchange resin (Bio-Rad) at a mobile phase flow-rate of 33 ml/h. The eluting solvents consist of 0.2 N buffers at pH 3.40,4.44 and 4.86 and a 0.35 N buffer at pH 5.86 (all of the buffers are sodium citrate). The separation of a number of pyridoxyl-... 

A modification of the pyridoxal—amino acid reaction (mentioned above) has been made for automatic analysis of amino acids by ligand-exchange chromatography [95]. This technique involves separation of the amino acids prior to fluorimetric reaction and determination. As the amino acids are eluted from the column, they are mixed with the pyridoxal-zinc(II) reagent to produce a highly fluorescent zinc chelate. Amounts of as low as 1 nmole of amino acid may be detected. The first reaction involved is the formation of the pyridoxyl-amino acid (Schiff base) as in Fig.4.46. The zinc then forms a chelate which probably has the structure shown in Fig. 4.48. 

Since the Schiff base formation is reversible, it should be reduced by sodium borohydride for the fixation of the label. The rate of the reduction of the Schiff base becomes slow as the number of the phosphate groups of the label increases. However, except for adenylate kinase, the NP -PL bound to the proteins were easily fixed by borohydride reduction. After reductive fixation, labeled proteins are cleaved by appropriate methods. The labeled lysine is cleaved by neither trypsin nor lysyl endopeptidase. There are at least three ways to detect the labeled peptide during isolation 1) use of radioactive reagent, 2) use of radioactive sodium borohydride for reduction of the Schiff base, and 3) use of fluorescence derived from the pyridoxyl moiety of the reagent (excitation at 295 nm and emission at 390 nm at acidic pH). The labeled lysyl residue is not positively identified in the amino acid sequence analysis. However, the presence of the label in the peptide isolated can be confirmed by the presence of pyridoxyl lysine in the amino acid analysis. 

Morino and Snell32 reduced holotryptophanase with NaBIL and isolated -pyridoxyl-lysine from the hydrolyzed protein. Subsequent isolation of the pyridoxyl-containing peptide50 and determination of its position in the total amino acid sequence of the enzyme led to identification of Lys-270, which forms a Schiff base with PLP. 

The reaction mixture contained l-5-HTP as substrate, pyridoxyl phosphate as a cofactor, pargylcine HQ, and the enzyme. The reaction was terminated by the addition of trichloroacetic acid (TCA), and after addition of the internal standard the reaction mixture was clarified by centrifugation. The sample was prepurifled on Amberlite, and the 5-HT eluted and injected onto the HPLC column for quantitation. The results obtained the following the incubation of 5-HTP with the homogenate are shown in Figure 9.2B. The formation of... 

In the presence of the cofactor pyridoxyl phosphate, Dopa decarboxylase catalyzes the decarboxylation of L-dopa to dopamine. This enzyme has been shown to be the same protein as 5-hydroxytryptophan decarboxylase, and both are referred to by the name aromatic L-amino acid decarboxylase (AADC). 

The incubation mixture contained ornithine, pyridoxyl phosphate, and the enzyme. After incubation for 1 hour, the reaction was terminated with perchloric acid. The precipitate was removed by centrifugation, the supernatant extracted with chloroform-methanol (2 1), and the aqueous layer applied to a CellexP column. The putrescine was eluted, reacted with fluorescamine, and quantitated by HPLC. 

Benzadox (and presumably, Irpexll) acts via metabolic conversion within plants to form amlno-oxyacetic acid, which Is a potent pyridoxyl phosphate-requiring enzyme Inhibitor (1901 and also a non-commercial synthetic herbicide (191. 1921. 

Proof that a lysine residue has been modified can be readily obtained because pyridoxyl derivatives of lysine possess characteristic white-blue fluorescence (Ronchi et al. 1969). In addition, they have a distinctive absorption maximum at 325 nm with of 9710 cm (Fisher et al. 1963). Finally, a radiochemical label can be introduced by reducing the pyridoxal-5-phosphate protein complex with tritium-labelled sodium borohydride. The peptide containing the derivatized lysine can therefore be detected either by fluorimetry, spectrophotometry or radiochemical techniques following routine procedures of proteolytic digestion and fractionation. Acid hydrolysis in 6 N HCl for 24 hr of peptides containing pyridoxal-5-phosphate lysine yields pyridoxyl-lysine since phosphate esters are readily hydrolyzed under these conditions. Pyridoxyl-lysine is eluted between lysine and histidine from a 55 cm column of Beckman 50 resin with 0.15 M citrate buffer pH 5.28. 

Deacetylation was accomplished by treating 258 mg of the above product for 40 min with 5 ml of 6 N HCl at 121 C. The resulting was dried under reduced pressure and dissolved in water. After adjusting the pH to 7.0 with aqueous ammonia, the solution was evaporated to dryness again. The residue was dissolved in a little water and applied to a 0.9 X 30 cm column of resin XE-64 which had been previously equilibrated with 1 M ammonium formate pH 4.0 and washed with 250 ml of water. The column was washed with 850 ml of water after application of the sample followed by 70 ml of 0.72 M acetic acid to remove unhydrolyzed material. The e-pyridoxyl lysine was eluted with 0.81 M acetic acid, and fractions containing the product were evaporated to dryness. On recrystallization of pyridoxyl-lysine from ethanol the yield was 110 mg of pale yellow crystals, m.p. 214-214.5°C. On paper chromatography in butanol pyridine acetic acid-HjO (30 20 6 24 v/v) Rf = 0.28 (Ronchi et al. 1969). 

In principle, pyridoxyl amino acids and other PLP derivatives can be used to treat bacteria, protozoans, and cancer cells evaluating their efficacy in depressing growth. This may lead to... 

Of the number of chromophoric derivatives of chiral amines for potential use in the establishment of their absolute configuration by BCD measurement only a few have proven to be generally useful. Of these, intensive investigation of the Af-salicylidene (Schiff base) derivatives of chiral primary amines, including unsubstituted and ring-substituted a- and S-arylalkylamines, a-amino acids, unsaturated and satnrated aliphatic and alicyclic amines, and amino sugars, has resulted in the formulation of the salicylidenamino chirality rule ° °. The application of this rule has recently been reviewed and has been successfully used for the establishment of absolute configuration of chiral primary amines in connection with other stereochemical studies. In related studies, the conformations of a series of pyridoxyl-L-a-amino acid Schiff bases were deduced from their CD spectra ... 

Pyridoxylamino acids exhibit spectral characteristics similar to those of pyridoxamine absorption maxima at 255 and 328 nm, and fluorescence emission at 400 nm. Fluorescence efficiency is pH-dependent, with maximum fluorescence at pH 5.28 (except for the histidine derivative, which shows maximum fluorescence at pH 12). Molar absorption coefficients and fluorescence efficiencies are, with few exceptions, the same for all pyridoxyl amino acids. Between 10 and lOOpmol of an amino acid can be determined in the effluent from an amino acid analyser [261]. 

The pyridoxyl residue increases the retention time, but most amino acid derivatives are eluted from the cation exchange column in the same order as the free amino acids. For continuous fluorescence measurement, one part of the effluent is mixed with 49 parts of sodium citrate buffer, pH 5.28. 

One of the advantages of the pyridoxal method is the possibility of using sodium borotritide (NaBT4), which is available with a specific activity of about 116 Ci mmol Thus, radioactively labelled pyridoxyl derivatives can be obtained, allowing the detection of about 0.1 pmol of an amine or amino acid. 

Vitamin is pyridoxal (ll.lOSf), pyridoxine (ll.lOSg) or pyridoxamine (ll.lOSh), all of which exist as their phosphate esters. This vitamin was first isolated in 1936. Pyridoxyl phosphate (ll.lOSi) is a versatile coenzyme used by all living organisms which participates in transamination (11.111) and (11.112), decarboxylation (11.113) and racemisation (11.114) reactions. It is the essential cofactor in amino acid metabolism. Virtually all enzymes which catalyse reactions of 2-amino acids utilise pyridoxyl phosphate as the coenzyme (11.111) through (11.114). 

Applications of tryptophan synthetase Tryptophan synthetase (EC 4.2.1.20) is a pyridoxal phosphate-dependent enzyme that, in the cell, catalyzes the a,/3-elimination of water from serine to form a pyridoxyl-bound a-aminoacrylate, which undergoes Michael addition of indole to form the named amino acid. This type of reaction has been used to prepare (5)-tryptophan isotopomers with a variety of labeling patterns by use of different labeled indoles and (5)-serines in yields of up to 98% based on indole and 92% based on (5)-serine. 

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