Pyridoxal-3-phosphate acetal

Histamine is synthesised by decarboxylation of histidine, its amino-acid precursor, by the specific enzyme histidine decarboxylase, which like glutaminic acid decarboxylase requires pyridoxal phosphate as co-factor. Histidine is a poor substrate for the L-amino-acid decarboxylase responsible for DA and NA synthesis. The synthesis of histamine in the brain can be increased by the administration of histidine, so its decarboxylase is presumably not saturated normally, but it can be inhibited by a fluoromethylhistidine. No high-affinity neuronal uptake has been demonstrated for histamine although after initial metabolism by histamine A-methyl transferase to 3-methylhistamine, it is deaminated by intraneuronal MAOb to 3-methylimidazole acetic acid (Fig. 13.4). A Ca +-dependent KCl-induced release of histamine has been demonstrated by microdialysis in the rat hypothalamus (Russell et al. 1990) but its overflow in some areas, such as the striatum, is neither increased by KCl nor reduced by tetradotoxin and probably comes from mast cells. 

L-/D-cvsteine. Hydrogen sulfide is produced from L-cysteine in a light-independent process that can be inhibited in vivo and in vitro by aminooxy acetic acid, an inhibitor of pyridoxal phosphate-dependent enzymes the hydrogen sulfide emitted in response to L-cysteine is directly derived from die L-cysteine fed ( 2 ). Therefore, hydrogen sulfide appears to be produced from L-cysteine by a pyridoxal phosphate-dependent, L-cysteine specific cysteine desulfhydrase. This conclusion is supported by the finding that in cucurbit... 

Tryptophanase catalyzes the conversion of tryptophan to indole and acetic acid. Pyridoxal phosphate is a required cofactor. The HPLC method developed to assay this activity involves the separation of the tryptophan from the indole. 

The borohydride reduction of Schiff base formed between a protein and pyridoxal phosphate should be carried out at a mildly acidic pH. Although sodium borohydride is more unstable in acidic solution, this disadvantage is offset by the exceptional reactivity of the Schiff base salts which are formed in mildly acidic solution (pH 4.S-6.5) (Schellen-berg 1963). Reductions have been carried out after the protein and pyridoxal-5-phosphate have been incubated at a pH of 7.5 which is then changed to 4.5 or 6.5 with acetic acid (Rippa et al. 1967 Dempsey and Christensen 1962 Piszkiewicz et al. 1970) or after initial incubation at pH 6.0 (Schnackerz and Noltmann 1971). The relative merit of either... 

TLC of vitamin Be compounds, on various layers in different solvents, was studied. The Rf values of pyri-doxine, pyridoxal, pyridoxamine, pyridoxal ethyl acetate, 4-pyridoxic acid, 4-pyridoxic acid lactone, pyridoxine phosphate, pyridoxal phosphate, and pyridoxamine phosphate were 0.62, 0.68, 0.12, 0.54, 0.91, 0.91, 0.95, 0.95, and 0.86, respectively, by TLC on silica gel HF254 with... 

The first step in the catabolism of most amino acids is the transfer of the o-amino group from the amino acid to a-ketoglutarate (tx-KG). This process is catalyzed by transaminase (aminotransferase) enzymes that require pyridoxal phosphate as a cofactor. The products of this reaction are glutamate (Glu) and the a-ketoacid analog of the amino acid destined for catabolic breakdown. For example, aspartate is converted to its a-keto analog, oxalo-acetate, by the action of aspartate transaminase (AST), which also produces Glu from a-KG. The transamination process is freely reversible, and the direction in which the reaction proceeds is dependent on the concentrations of the reactants and products. These reactions do not effect a net removal of amino nitrogen the amino group is only transferred from one amino acid to another. 

Figure 4 Absorbance spectrum for pyridoxal phosphate, alone and 1n the presence of aminooxy acetate(l), propionate(2), butyrate(3) and valerate(4).

Our laboratory has studied the stereochemistry of methyl group formation in a number of a, 0 elimination reactions of amino acids catalyzed by pyridoxal phosphate enzymes. The reactions include the conversions of L-serine to pyruvate with tryptophan synthase 02 protein (78) and tryptophanase (79), of L-serine and l-tyrosine with tyrosine phenol-lyase (80), and l-cystine with S-alkylcysteine lyase (81). In the latter study, the stereospecific isotopically labeled L-cystines were obtained enzymatically by incubation of L-serines appropriately labeled in the 3-position with the enzyme O-acetyl serine sulfhy-drase (82). The serines tritiated in the 3-position were prepared enzymatically starting from [l-3H]glucose and [l-3H]mannose by a sequence of reactions of known stereochemistry (81). The cysteines were then incubated with 5-alkyl-cysteine lyase in 2H20 as outlined in Scheme 19. The pyruvate was trapped as lactate, which was oxidized with K2Cr202 to acetate for analysis. Similarly, Cheung and Walsh (71) examined the conversion of D-serine to pyruvate with... 

Cysteine synthesis in bacteria proceeds via a-aminoacrylic acid bound to the enzyme as a Schiff base with pyridoxal phosphate (Cook and Wedding, 1976). Partially purified cysteine synthase from spinach (Schmidt, 1977a) and Chlorella (Schmidt, 1977b) catalyzes an exchange of sulfide into cysteine, consistent with the above mechanism. The exchange of acetate into OAS that would be expected due to formation of the proposed enzyme intermediate was not tested. 

Karrer and Viscontini synthesized the acetal of pyridoxal-3-phosphate and claimed that it had codecarboxylase activity but could not reactivate a transaminase system. In the meantime, Gunsalus presented evidence that the active synthetic product was not the 3-phosphate (and by elimination must be the 5-phosphate). This discrepancy was resolved when the two products were compared simultaneously on the same test system. The activity of the 3-phosphate was so low compared to the active pyridoxal phosphate that the 3-phosphate was definitely ruled out as the active coenzyme. In a recent series of papers,the structure of pyridoxal phosphate is definitely established as pyridoxal-5-phosphate and that of pyridoxamine-phosphate as the 5 phosphate. 

ATPase with pyridoxal-5 -phosphate [341-344], adenosine-5 -triphosphopyridoxal [99], 2,4,6-trinitrobenzene sulfonate [344-346], fluorescamine [347,348], methylbenzi-midate [349], acetic anhydride and maleic anhydride [344], and o-phthalaldehyde [350],... 

Vitamins of group B were analysed in different forms [530]. Isopropylidene derivatives showed selectivity of the chromatographic separation which was caused by even minor structural differences. Several compounds from the pyridoxine group can be analysed after their conversion into acetates acetylation followed by GC also appeared suitable for three vitamins and 4-pyridoxic lactone. TMS derivatives were recommended for GC separation of the phosphate form of vitamins. When treated with BSTFA—pyridine (1 1) at 60°C for 15 min, biotin provides a completely silylated derivative, which was analysed on a column packed with 3% of OV-17 [531 ]. 

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... 

Colman and Frieden (108) demonstrated in 1966 that acetylation of one amino group per subunit with acetic anhydride produces 80% inactivation. More extensive acetylation alters the degree of polymerization and certain kinetic parameters (276). Almost simultaneously, Anderson et al. (277) reported the reversible inhibition of GDH by pyridoxal 5 -phosphate and certain other aromatic aldehydes. The inhibition was attributed to formation of a Schilf base since reduction with NaBH4 results in irreversible inactivation. It was estimated that approximately one residue of -pyridoxyllysine had been formed per subunit. In 1969, Holbrook and Jeckel (278) inactivated the enzyme by reaction with a substituted maleimide and, subsequently, obtained the partial sequence of a tryptic peptide containing a modified lysine residue (Fig. 7). 

DPTA = diethylene triamine penta-acetic acid BMA = 5,8-bis(carboxymethyl)-ll-[2-(methylamino)-2-oxoethyl]-3-oxo-2,5,841-tetra-azatridecan-13-oic acid BMEA = N,N -bis[methoxyethylamide] BOPTA = benzyloxypropionic tetra-acetic-acid DPCP = N,N -bis[pyridoxal-5-phosphate]-trans-l,2-cyclohexyldiamine-N , N -diacetic acid EOB-DTPA= ethoxybenzyldiethylene triamine penta-acetic acid BT-D03A= 10-[2,3-dihydroxy-l-hydroxymethylpropyl]-l,4,7,10-tetra-azacyclododecane-l,4/7-triacetic acid HP-D03A= 10-[2-hydroxypropyl]-l,4/7-tetra-azacyclododecane-l,4/7-triacetic acid DOTA= 1,4,7, 10-tetra-azacyclododecane-N,N, N",N "-tetra-acetic acid. 

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