Pyridoxyl-5-phosphate

Members of the NifS/IscS family are pyridoxyl-5 -phosphate dependent proteins that deliver inorganic sulfide during assembly of the [2Fe-2S] cluster on the IscU scaffold protein. While [2Fe-2S] cluster reconstitution of apo Fe-S proteins can often be achieved in vitro by addition of excess iron and sulfide in... 

The final reaction in the biosynthesis of threonine involves a /8-y rearrangement and the loss of phosphate from O-phosphohomoserine (Fig. 2). Threonine synthases have been isolated from Lemna (Schnyder et al., 1975) radish, sugarbeet (Madison and Thompson, 1975), peas (Schnyder et al., 1975 Thoen et al., 1978b), and barley (Aames, 1978). None of these enzymes has been extensively characterized but a requirement for pyridoxyl-5 -phosphate was demonstrated after partial purification of the barley and pea enzymes. Unlike several other enzymes associated with threonine synthesis, the activity of threonine synthase was not stimulated by monovalent cations. However, all of the plant enzymes are strongly activated by 5-adeno-sylmethionine (Section III,B,5). 

AOPP and AOA inhibit transaminase enzymes (39, 44) and other pyridoxylphosphate-dependent enzymes, presumably by interference with the carbonyl group of pyridoxyl phosphate (45). They apparently inhibit PAL by interaction with the carbonyl-like group involved in catalysis by PAL (36). AOA is not an effective PAL inhibitor for in vivo studies because of its lack of specificity that results in a relatively high degree of phytotoxicity (e.g. 

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. 

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. 

Depressed synthesis or the total absence of glycine causes the accumulation of 5-phospho-/J-D-ribosylamine, the other substrate of the enzyme phosphoribosylglycinamide synthase. Reaction of pyridoxyl phosphate with alanine ... 

The application of the biocatalysts in this overview is limited to these stable enzymes, which do not need cofactors, such as the various hydrolytic enzymes, some lyases, transferases and isomerases. In addition to these groups, oxidoreductases, which demand NAD or NADP as cofactors, some pyridoxyl-phosphate dependent lyases with simple systems for cofactor regeration and finally, various aldolases in combination with L-glycerol- phosphate oxidase and catalase are useable to some extent in cell-free form. 

Pyridone Pyridone-2 2(1 H)-Pyridone a-Pyridone. See 1-Hydroxy-2-pyridine Pyridoxaldehyde phosphate Pyridoxal, 5-(dihydrogen phosphate) Pyridoxal monophosphate Pyridoxal P Pyridoxal phosphate. See Pyridoxal 5-phosphate Pyridoxal 5-phosphate CAS 54-47-7 41468-25-1 (monohydrate) EINECS/ELINCS 200-208-3 Synonyms Codecarboxylase 3-Hydroxy-2-methyl-5-[(phosphonooxy) methyl]-4-pyridinecarboxaldehyde Phosphopyridoxal Phosphoridoxal coenzyme 4-Pyridinecarboxaldehyde, 3-hydroxy-2-methyl-5-[(phosphonooxy) methyl]-Pyridoxaldehyde phosphate Pyridoxal, 5-(dihydrogen phosphate) Pyridoxal monophosphate Pyridoxal P Pyridoxal phosphate Pyridoxyl phosphate Classification Organic compd. 

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). 

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-... 

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. 

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. 

Authentic pyridoxyl-lysine has been prepared by Schnackerz and Noltmann (1971) by borohydride reduction of a mixture of poly-L-lysine hydrochloride (20 mg) and 0.38 moles of pyridoxal, which had been allowed to stand for 10 min at 0°C in 50 mM sodium phosphate (pH 6.0). Low molecular weight impurities were removed by dialysis against 50 mM sodium acetate buffer (pH 6.0). After dialysis the resulting product can be hydrolyzed in 6 N HCl to yield pyridoxyl-lysine and some lysine. The latter contaminant can be minimized by... 

Phosphonylation of a -3-(9-isopropylidene-a -pyridoxyl chloride via a Michaelis-Becker reaction, followed by deprotection with 1 M HCl and oxidation of the resulting primary alcohol with MnOj, produces diethyl (4-fonnyl-3-hydroxy-2-methyl-5-pyridyl)methylphosphonate (Scheme 5.38), an analogue of pyridoxal 5 -phosphate whose 5-position side chain has been replaced by a phospho-nomethyl group. The alcohol oxidation step can be accomplished with a wide range of reagents, such as activated MnOj in CHCI3 at room temperature (53%), PCC in CH2CI2 (83-86%), or the Swem reaction (>95%). ... 

A phosphonate analogue (8) of pyridoxal 5 -phosphate (9) which contains a 5 -phosphonomethyl group has been prepared by a Michaelis-Becker reaction from the protected pyridoxyl chloride (10). Both (8) and its monoethyl ester... 

Energy transfer has also been widely useful for measurements of protein folding. One exan le is fcr the protein serine hydroxymethyltransferase. This protein typically has three tryptophan residues, at positions 16, 183. and 385. The acceptor was a pyridoxyl S -phosphate (PyP) residue covalently linked to lysine-229. One expects protein folding or unfolding to affect the distance from each of the tryptophan residues to PyP, and thus one expects the extent of RET to be differs for the n ve and folded proteins. 

Pyridoxal and pyridoxal 5-phosphate are the only naturally occurring compounds which have been used as fluorescent labels. These compounds form Schiff bases with primary amino groups. The reduction of the C=N bond with sodium borohydride (NaBH4) leads to the formation of fluorescent pyridoxyl derivatives [260,261],... 

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