Pyridoxamine 5-phosphate

Figure 11.3 Regulation of GAD during the synthesis of GABA. Active GAD (GAD-PLP) combines with glutamate (1) to form a complex (GAD-PLP-GLU). After decarboxylation (2) this yields GABA and GAD-PLP (3). The intermediate product (GAD-INT) can undergo an alternative reaction (4) to produce succinic semialdehyde (SSA) and pyridoxamine-5 -phosphate (PMP). PMP dissociates from GAD (5) leaving inactive enz5mie, which requires additional PLP to be reactivated (6), a process that is affected by ATP and inorganic phosphate...

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... 

Hydrolysis of the new imine then allows formation of a ketone as part of an a-keto acid, and an amine which is the previously mentioned pyridoxamine 5 -phosphate. Since this imine is the product from an amine and a ketone, it is termed a ketimine. These reactions are reversible in nature, allowing amino acids to be converted into keto acids, and keto acids to be converted into amino acids (see Section 15.6). 

Metabolites that contain pyridoxol (2-methyl-3-hydroxy-4,5-di[hydroxymethyl]pyridine), the water-soluble vitamin B6. Phosphorylated forms of pyridoxal (the aldehyde form) and pyridoxamine (the amino form), known respectively as pyridoxal 5-phosphate (PLP) and pyridoxamine 5-phosphate (PMP), are tightly-bound coenzymes. 

Marafie, et al. Complexes of vitamin B6 XX equilibrium and mechanistic studies of the reaction of pyridoxal-5 -phosphate with pyridoxamine-5 -phosphate in the presence of copper(II). J Inorg Biochem 1989 37 7-16. 

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 conversion includes at least three enzymic reactions.169-171 In the first stage, which requires pyridoxamine 5 -phosphate as a cofactor,171,172 dehydration of 7b occurs through intermediate formation of the Schiff base.173 Reduction of the resulting, unsaturated derivative with NADPH, the mechanism of which is not completely clear,174 leads to CDP-3,6-dideoxy-D-eryf/iro-hexos-4-ulose,169 and, in the third stage, further reduction of the latter at C-4 of the hexosyl group produces the derivatives of paratose or abequose the stereochemistry of the reaction is determined by the source of the enzyme.168 The tyvelose derivative is formed as a result of enzymic epimerization at C-2 of the hexosyl group in CDP-paratose.175... 

The full series of intermediates in a transamination is shown in figure 10.5a. After protonation at the aldimine carbon of pyridoxal-5 -phosphate (step 3), hydrolysis (step 4) forms an a-keto acid and pyridoxamine-5 -phosphate. The reverse of this sequence with a second a-keto acid (steps 5 through 8) completes the transamination reaction. 

Figure 5.8 Separation of eleven water-soluble vitamins by MECC. Peaks 1, pyridoxamine 2, nicotinamide 3 pyridoxal 4, vitamin B6 5, vitamin B2 6, vitamin B12 7, vitamin B2 phosphate 8, pyridoxamine 5 -phosphate 9, niacin 10, vitamin Bi 11, pyridoxal 5 -phosphate. Conditions buffer, 50 mM SDS in 20 mAf phosphate-borate buffer, pH 9.0 applied voltage, 20 kV detection, UV absorbance at 210 nm. (Reprinted from Ref. 20 with permission.)...

Amino acids NAD(P)H Pyridoxamine 5 -phosphate Pyruvate Amines Pyridoxal 5-phosphate dependent enzymes Dehydrogenases Transaminases Pyridoxal 5-phosphate dependent enzymes Amino acid decarboxylases... 

Three enzymes play an active role in the metabolism of vitamin B6 in human erythrocytes. Pyridoxal kinase uses ATP to phosphorylate pyridoxine, pyri-doxamine, and pyridoxal. Pyridoxamine oxidase oxidizes pyridoxamine-5 -phosphate and pyridoxine-5 -phosphate to pyridoxal-5 -phosphate. The phosphatase activity produces pyridoxal from pyridoxal-5 -phosphate. The assay of the three enzymes required separation of the semicarbazone derivatives of pyridoxal-5 -phosphate and pyridoxal. The mobile phase used by Ubbink and Schnell (1988) contained 2.5% acetonitrile. Detection was by fluorescence. 

Pyridoxal-5 phosphate (P-5 -P) and its amino analogue, pyridoxamine-5 -phosphate, function as coenzymes in the amino-transfer reactions. The P-5 -P is bound to the apoen-zyme and serves as a true prosthetic group. The P-5 -P bound to the apoenzyme accepts the amino group from the first substrate, aspartate or alanine, to form enzyme-bound pyridoxamine-5 -phosphate and the first reaction product, oxaloacetate or pyruvate, respectively. The coenzyme in amino form then transfers its amino group to the second substrate, 2-oxoglutarate, to form the second product, glutamate. P-5 -P is thus regenerated. 

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). 

The final step in this pathway is the oxidation of PNP to PLP and is carried out by PdxH. Tbe recombinant enzyme from E. coli has been studied in vitro and is a 51 kDa homodimer that utilizes flavin mononucleotide (FMN) as a cofactor. PdxH can use either PNP or pyridoxamine 5 -phosphate (PMP) as a substrate with a of 2 and 105 pM and cat of 0.8 and 1.7 s for PNP and PMP, respectively. The structures of the enzyme from E. coli as well as homologues from Mycobacterium tuberculosis and humans have been solved. The E. coli enzyme with PLP and FMN bound is shown in Figure 6. PdxH is involved in both the biosynthetic and the salvage pathways and is further discussed in a section describing the transport, salvage, and interconversion of the various forms of vitamin Bg. 

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