Cofactors pyridoxal phosphate

In nature, aminotransferases participate in a number of metabolic pathways [4[. They catalyze the transfer of an amino group originating from an amino acid donor to a 2-ketoacid acceptor by a simple mechanism. First, an amino group from the donor is transferred to the cofactor pyridoxal phosphate with formation of a 2-keto add and an enzyme-bound pyridoxamine phosphate intermediate. Second, this intermediate transfers the amino group to the 2-keto add acceptor. The readion is reversible, shows ping-pong kinetics, and has been used industrially in the production ofamino acids [69]. It can be driven in one direction by the appropriate choice of conditions (e.g. substrate concentration). Some of the aminotransferases accept simple amines instead of amino acids as amine donors, and highly enantioselective cases have been reported [70]. 

These enzymes invariably involve a cofactor, pyridoxal phosphate (vitamin B6). In addition, pyridoxal phosphate is also required for most decarboxylations, racemizations, or elimination reactions in which an amino acid is a substrate. Pyridoxal phosphate is not involved in decarboxylations in which the substrate is not an amino acid. So if a question... 

The amino acid and nucleotide biosynthetic pathways make repeated use of the biological cofactors pyridoxal phosphate, tetrahydrofolate, and A-adenosylmethionine. Pyridoxal phosphate is required for transamination reactions involving glutamate and for other amino acid transformations. One-carbon transfers require S-adenosyhnethionine and tetrahydrofolate. Glutamine amidotransferases catalyze reactions that incorporate nitrogen derived from glutamine. 

Aminotransferases operate in both directions. Their mechanism uses the cofactor pyridoxal phosphate to form Schiff bases with amino groups, as shown in Figure 4-3. 

The glycine-dependent aldolases contain a cofactor pyridoxal phosphate (PLP). Binding of glycine to it as an imine enables the deprotonation necessary for the carbon-carbon bond forming reaction, with pyridine acting as an electron sink. The subsequent 100% atom efficient reaction with an aldehyde establishes the new bond and two new stereocenters (Scheme 5.30). Of all the glycine-dependent aldolases only L-threonine aldolase (LTA) is commonly used [40, 43, 52]. 

Vitamin B or pyridoxine participates in over 100 enzymatic reactions as the cofactor, pyridoxal phosphate (PLP). It exists in three forms the alcohol, the amine, or the aldehyde. Pyridoxal phosphate is an essential cofactor for enzymes involved in the synthesis of many neurotransmitters. 

The reactions catalyzed by aminotransferases arc called transaminahon reactions. It might he not that in these reactions the amino group being transferred initially is transferred to the cofactor pyridoxal phosphate, resulting in its conversion to pyridoxamine phosphate. In the second half of the reaction, the amino group residing on the cofactor is transferred to the keto acid cosubslrate, thus regenerating the cofactor in the pyridoxal phosphate form. As stated earlier, the cofactor remains bo Lind to the enzyme when it occurs as the pyridoxal phosphate and pyridoxamine phosphate forms. 

Oxidative deamination of alanine requires the cofactor pyridoxal phosphate and yields pyruvate as product. 

Scheme 4.36 The antiprotozoal drug Eflornithine acts by inhibiting ornithine decarboxylase (OD) box), forming covalent bonds to the enzyme and to the cofactor pyridoxal phosphate (PLP ...

For continuous production of L-p-fluorophenylalanine, a typical set of operating conditions is shown in Table 12.7-2. L-Aspartate is used at a 10 % molar excess to the starting 2-ketoacid. The cofactor pyridoxal phosphate is added to the reaction mixture to achieve a final concentration of 0.1 mM. The initial pH of the feed solution is 7.2. Mg2+ ion was used to accelerate the decarboxylation of oxaloacetate to pyruvate. The reaction was maintained with a temperature range of37-40 °C. Under these conditions using an immobilized broad-range aminotransferase, the volumetric productivity of the reactor for the production of L-phenylalanine at 85% conversion was 20 gL 1h 1. 

Plasma AST measurements are a useful adjunct to ALT, and increased AST can be an indication of mitochondrial and cytoplasmic injury, although this enzyme is less specific for hepatotoxicity compared to ALT. Sustained increases of the plasma aminotransferase levels can indicate a progressive injury, but the aminotransferases may not be elevated during acute necrosis if the timing of the sample collection has allowed the increased circulating plasma enzymes to be cleared. Plasma ALT and AST may be decreased when the enzyme cofactor pyridoxal phosphate is reduced in vivo (Dhami et al. 1979 also see Chapter 2). 

Amino acid metabolism requires the participation of three important cofactors. Pyridoxal phosphate is the quintessential coenzyme of amino acid metabolism (see Chapter 38). All amino acid reactions requiring pyridoxal phosphate occur with the amino group of the amino acid covalently bound to the aldehyde carbon of the coenzyme (Fig. 39.3). The pyridoxal phosphate then pulls electrons away from the bonds around the a-carbon. The result is transamination, deamination, decarboxylation, P-elimination, racemization, and -elimination, depending on which enzyme and amino acid are involved. 

In adults, a genetic deficiency of cystathionase causes cystathionuria. Individuals with a genetically normal cystathionase can also develop cystathionuria from a dietary deficiency of pyridoxine (vitamin B6), because cystathionase requires the cofactor pyridoxal phosphate. No characteristic clinical abnormalities have been observed in individuals with cystathionase deficiency, and it is probably a benign disorder. 

Some results indicate that different attempts of FucA immobilization by covalent attachment provoked severe enzyme inactivation (Fessner and Walter 1997). FucA and DERA from E. coli and SHMT from Streptococcus thermophilus have been immobilized by multipoint covalent attachment to glyoxyl-agarose. Although this immobilization method had been very successful with many different enzymes (Guisdn et al. 1993), results obtained with these aldolases were dissimilar. For FucA, in spite of an immobilization yield of 80-90%, enzyme inactivation occurred during immobilization process and only 10-20% of activity was retained (Suau et al. 2005). On the other hand, SHMT immobilization yield was 100%, but the immobilized activity was lost during the sodium borohydride reduction step, probably due to the reduction of the Schiff base established between the cofactor (pyridoxal phosphate) and the aldolase. Finally, 100% of immobilization yield and 65% of retained activity in the immobilized derivative was achieved with DERA. 

Vitamin B6 is one of the most versatile enzyme cofactors. Pyridoxal phosphate-containing proteins are found in each lUB enzyme category except ligases (category 6). [Tong and Davis (1995) reported that 2-amino-3-ketobutyrate-CoA ligase is a pyridoxal phosphate enzyme. However, the... 

Butler, P. E., Cookson, E. J., and Beynon, R. J. (1985). The turnover of skeletal musde glycogen phosphorylase studied using the cofactor, pyridoxal phosphate, as a specific label. Biochim. Biophys. AcU> 847, 316-323. 

Although yeast cells were considered to incorporate up to 50% of wort amino acids directly into protein [62], analysis of the utilization of and labelled amino acids by brewers yeast show that negligible assimilation of complete amino acid occurs [63]. Thus, when amino acids enter the cell their amino groups are removed by a transaminase system and their carbon skeletons assimilated. Transaminases catalyse readily reversible reactions dependent upon the presence of the cofactor pyridoxal phosphate. The general mechanism of the reaction is depicted in Fig. 17.14. 

Fig. 17.14 Participation of the cofactor pyridoxal phosphate in the process of transamination.

One of the pathways to propanoyl-CoA is from catabolism of the amino acid threonine (Chapter 12). Thus, threonine (threonine dehydratase, EC 4.3.1.19, cofactor pyridoxal phosphate) undergoes deamination to give 2-oxobutanoate (a-ketobutyrate) as shown below. Then, 2-oxobutanoate (a-ketobutyrate) undergoes decarboxylation (perhaps as shown in Scheme 11.30) with formation of propanoyl dihydro-lipoamide in a (cofactor) thiamine diphosphate mediated step. Finally, as in Scheme 11.31, propanoyl-CoA is formed. An alternative pathway uses aferrodoxin to effect the decarboxylation of 2-oxobutanoate (a-ketobutyrate) Ferredoxins are small proteins containing iron and sulfur atoms in iron-sulfur clusters. 

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