Pyridoxal and Pyridoxamine

Goldberger and Lillie in 1926 found that rats fed certain nutritionally deficient diets developed dermatitis acrodynia, a skin disorder characterized by edema and lesions of the ears, paws, nose, and tail. Szent-Gyorgyi later found that a factor he had isolated prevented these skin lesions in the rat. He proposed the name vitamin Bg for his factor. Pyridoxine, a form of this vitamin found in plants (and the form of Bg sold commercially), was isolated in 1938 by three research groups working independendy. Pyridoxal and pyridoxamine, the forms that predominate in animals, were... 

Figure 3. Addition, of the GOT coenzymes pyridoxal and pyridoxamine phosphates in concentrations up to 200 /tg/ml lias no effect on human serum GOT but activates bu 45% the pig heart GOT activity of VersatoUE, a commercial reference serum...

Pyridoxal Phosphate.—Analogues of pyridoxal and pyridoxamine 5 -phosphates have frequently been used to probe the size and shape of the active sites of a number of enzymes. For example, the apoenzyme of a tryptophanase from Bacillus alvei will bind pyridoxal 5 -phosphate as well as the 2-nor, 2 -methyl, 2 -hydroxy, 6-methyl, and A-oxide analogues.27 No analogue that has been modified at C-4 binds to the enzyme, confirming the absolute requirement for Schiff-base formation between the... 

Pyridoxal and Pyridoxamine Amino Add Chimera Containing Peptides... 

Work in the Imperiali laboratory has also focused on exploring the ability of minimal peptide scaffolds to augment the rate of coenzyme-mediated transaminations [22-25]. To accomplish this, a strategy has been developed in which the core functionality of the coenzyme is incorporated as an integral constituent of an unnatural coenzyme amino acid chimera construct. Thus, non-cova-lent binding of the coenzyme to the peptide or protein scaffold is unnecessary. Both the pyridoxal and pyridoxamine analogs have been synthesized in a form competent for Fmoc-based solid phase peptide synthesis (SPPS) (Fig. 7) [23,24]. 

The terminology vitamin Bg covers a number of structurally related compounds, including pyridoxal and pyridoxamine and their 5 -phosphates. Pyridoxal 5 -phosphate (PLP), in particular, acts as a coenzyme for a large number of important enzymic reactions, especially those involved in amino acid metabolism. We shall meet some of these in more detail later, e.g. transamination (see Section 15.6) and amino acid decarboxylation (see Section 15.7), but it is worth noting at this point that the biological role of PLP is absolutely dependent upon imine formation and hydrolysis. Vitamin Bg deficiency may lead to anaemia, weakness, eye, mouth, and nose lesions, and neurological changes. 

Vitamin B6 is a collective term for pyridoxine, pyridoxal, and pyridox amine, all derivatives of pyridine. They differ only in the nature of the functional group attached to the ring (Figure 28.10). Pyridoxine occurs primarily in plants, whereas pyridoxal and pyridoxamine are found in foods obtained from animals. All three compounds can serve as precur sors of the biologically active coenzyme, pyridoxal phosphate. Pyridoxal phosphate functions as a coenzyme for a large number of enzymes, par ticularly those that catalyze reactions involving amino acids. 

Biotin-Containing Enzymes. 2. The Mechanism of Biotin Action 738 Box 14-C The Vitamin B6 Family Pyridoxine, Pyridoxal, and Pyridoxamine... 

In 1944, Esmond Snell reported the nonenzymatic conversion of pyridoxal into pyridoxamine (Box 14-C) by heating with glutamate. He recognized that this was also transamination and proposed that pyridoxal might be a part of a coenzyme needed for aminotransferases and that these enzymes might act via two halfreactions that interconverted pyridoxal and pyridoxamine (Eq. 14-25). The hypothesis was soon verified and the coenzyme was identified as pyridoxal 5 -phos-phate or pyridoxamine 5 -phosphate (Fig. 14-5).144/145... 

The binding of a symmetric chromophore to a protein or nucleic acid often induces CD in that chromophore. For example, the bands of enzyme-bound pyridoxal and pyridoxamine phosphates shown in Fig. 14-9 are positively dichroic in CD, but the band of the quinonoid intermediate at 20,400 cm-1 (490 nm) displays negative CD. When "transimination" occurs to form a substrate Schiff base (Eq. 14-26), the CD is greatly diminished. While the coenzyme ring is known to change its orientation (Eq. 14-39 Fig. 14-10), it is not obvious how the change in environment is related to the change in CD. 

In 1934, Gyorgy cured a dermatitis in rats (not due to vitamins Bj or B2) with a yeast extract factor, In 1938, Lepkovsky isolated a similar factor from nee bran extract. In that same year. Keresztesy and Stevens isolated and crystallized pure (, from rice polishings. Also, in the same year, Kohn, Wendt, and Westphal synthesized pyridoxine and gave pyridoxine its present name. In the following year (1939). Stiller, Keresztesy, and Stevens established the structure of the vitamin, In 194 5, Snell observed pyridoxal and pyridoxamine. The recognition of and establishment of B5 requirements in humans was not achieved until 1953, by Snyderman et al. 

Pyridoxine, pyridoxal, and pyridoxamine, which occur in foodstuffs, are collectively known as vitamin Bg. In the body, all three are converted to pyridoxal phosphate which is the coenzyme for amino-acid decarboxylase and for transaminase. The structures of the three active forms of vitamin Bg and the pyridoxal phosphate, are shown below (55). 

Vitamin B6 occurs in animal tissues in the form of pyridoxal and pyridoxamine or as their phosphates. Pyridoxine occurs in plant products. 

Tissue uptake of vitamin Be is again by carrier-mediated diffusion of pyridoxal (and other unphosphorylated vitamers), followed by metabolic trapping by phosphorylation. Circulating pyridoxal and pyridoxamine phosphates are hydrolyzed by extracellular alkaline phosphatase. All tissues have pyridoxine kinase activity, but pyridoxine phosphate oxidase is found mainly in the liver, kidney, and brain. 

Vitamin B Three substances are classed under the term pyridoxine or adermine pyridoxol, pyridoxal and pyridoxamine. Pyridoxine was isolated by various study groups in 1938. Its structure was described by Folkers and Kuhn in 1939. Pyridoxal and pyridoxamine were discovered by Snell in 1942. Pyridoxal phosphate and pyridoxamine phosphate are biologically active substances. Intestinal absorption of Bg is dose-dependent and not limited. In alcoholism, a deficiency of vitamin Bg is encountered in 20—30% of cases, whereas the respective percentage is 50—70% in alcoholic cirrhosis. Vitamin Bg is an important coenzyme for transaminases, which transfer amino groups from amino adds to keto acids. In this way, biochemical pathways between the dtiic acid cycle and carbohydrate and amino acid metabolisms are created. (104)... 

Pyridoxine Hydrochloride, USP. Pyridoxine hydrochloride.. S-hydroxy-6-methyl-3.4-pyridinedimethanol hydrochloride. vitamin B, hydrochloride, rat antidermatitis factor. is a white, odorless, crystalline substance that is soluble l .S in water and 1 100 in alcohol and in.soluble in ether. It is relatively. stable to light and air in the solid form and in acid solutions at a pH no greater than S. at which pH it can be autoclaved at IS pounds at I20°C for 20 to 30 minutes. Pyridoxine is unstable when irradiated in aqueous solution.s at pH 6.8 or above. It is oxidized readily by hydrogen peroxide and other oxidizing agents. Pyridoxine is as stable in mixed vitamin preparations as riboflavin and nicotinic acid. A 1% aqueous solution has a pH of 3. The pK i values fur pyridoxine. pyridoxal. and pyridoxamine are S.OO. 4.22. and 3.40. respectively, and their pK 2 values are 8.96. 8.68. and 8.05. respectively. 

L-Amino acid transaminases are ubiquitous in nature and are involved, be it directly or indirectly, in the biosynthesis of most natural amino acids. All three common types of the enzyme, aspartate, aromatic, and branched chain transaminases require pyridoxal 5 -phosphate as cofactor, covalently bound to the enzyme through the formation of a Schiff base with the e-amino group of a lysine side chain. The reaction mechanism is well understood, with the enzyme shuttling between pyridoxal and pyridoxamine forms [39]. With broad substrate specificity and no requirement for external cofactor regeneration, transaminases have appropriate characteristics to function as commercial biocatalysts. The overall transformation is comprised of the transfer of an amino group from a donor, usually aspartic or glutamic acids, to an a-keto acid (Scheme 15). In most cases, the equilibrium constant is approximately 1. 

Vitamers are chemically similar substances that have a qualitatively similar vitamin activity. Thus, vitamin D refers to ergocalciferol (Da) and cholecalciferol (D3) and sometimes to their 25-hydroxy- and 1,25-dihydroxy derivatives (Chapter 37). Similarly, pyridoxine (pyri-doxol), pyridoxal, and pyridoxamine are vitamin Be vitamers, riboflavin is the active form of vitamin Ba and cobalamin is vitamin Bia- The members of a particular vitamin family are functionally interchangeable and protect against deficiency symptoms for that vitamin. A vitamin and its corresponding deficiency disease are related as follows ... 

For most enzymes, the coenzyme form of pyridoxine is pyridoxal 5-phosphate. The transaminases can use pyridoxamine 5-phosphate because they interconvert pyridoxal and pyridoxamine during their activity. The three vitamers are readily converted to the active form (Figure 38-15). 

ORD measurements in the range 300—600 nm of the native enzyme showed Cotton effects due to the coenzymes (pyridoxal and pyridoxamine phosphate). These effects were absent in the apoenzyme, in the coenzymes alone, and at high substrate concentrations (357). CD data obtained for this enzyme were transformed to ORD curves, which were in good agreement with ORD data measured previously (358). 

Pyridoxic Acid, 3-Hydroxy-5-(hydroxymethyl)-2-meihyl-4-pyridinecar boxy lie acid 3-hydroxy-5-(hydroxy-methyl)-2-methylisonicotinic acid 2 -methyl-3 -hydroxy -4-carboxy-5-hydroxymethylpyridine. CgHjNO, mol wt 183,16. C 52.46%, H 4.95%, N 7.65%, O 34.94%. Occurs in urine. It is the chief metabolic product of pyridoxine pyridoxal, and pyridoxamine. Isoln from human urine Singal, Sydenstricker, Science 78, 545 (1941). Isoln and synthesis Huff, Perlzweig, J. Biol Chem ]S5, 345 (1944). 

Pyridoxine (peer-ih-DOCK-seen) is also known as 3-Hydroxy-4,5-bis(hydroxymethyl)- -methyl pyridine 3-hydroxy-4,5-dime-thylol-2-methylpyridine and vitamin B6. It is a white, odorless, crystalline compound with a slightly bitter taste. The term pyridoxine is also used as a generic term for three compounds with biological activity classified under the term Vitamin Bfc. The three compounds are pyridoxine, pyridoxal, and pyridoxamine. Pyridoxine is usually produced commercially as the hydrochloride, CH3CSHN(0H)(CH20H)2-HC1, which has somewhat different physical characteristics from pyridoxine itself. 

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