Pyridoxal phosphate regeneration

Cofactor regeneration is not only a task when using oxidoredurtases but also for some representatives from other enzyme classes, for example, transferases and lyases. Transaminases require pyridoxal phosphate and transform a ketone into the chiral amine on the expense of a donor amine that is the cosubstrate needed in stoichiometric amount [17]. A representative example for pyridoxal phosphate regeneration is shown in Scheme 2.5, exemplified for the use of isopropylamine as an amine donor. This reagent is especially useful for two reasons. First, it is a small (and thus atom economical), cheap, and readily available amine. 

FIGURE 14.22 Glutamate aspartate aminotransferase, an enzyme conforming to a double-displacement bisnbstrate mechanism. Glutamate aspartate aminotransferase is a pyridoxal phosphate-dependent enzyme. The pyridoxal serves as the —NH, acceptor from glntamate to form pyridoxamine. Pyridoxamine is then the amino donor to oxaloacetate to form asparate and regenerate the pyridoxal coenzyme form. (The pyridoxamine enzyme is the E form.)... 

Pyridoxal phosphate (aldimine form, on regenerated enzyme)... 

There is an important biochemical counterpart of the deamination reaction that utilizes pyridoxal phosphate, 7, as the aldehyde. Each step in the sequence is catalyzed by a specific enzyme. The a-amino group of the amino acid combines with 7 and is converted to a keto acid. The resulting pyridoxamine then reacts to form an imine with a different a-keto acid, resulting in formation of a new a-amino acid and regenerating 7. The overall process is shown in Equation 25-6 and is called transamination. It is a key part of the process whereby amino acids are metabolized. 

The amino acid is then hydrolyzed to form an a-keto acid and pyridoxamine phosphate, the a-amino group having been temporarily transferred from the amino acid substrate on to pyridoxal phosphate (Fig. 5). These steps constitute one half of the overall transamination reaction. The second half occurs by a reversal of the above reactions with a second a-keto acid reacting with the pyridoxamine phosphate to yield a second amino acid and regenerate the enzyme-pyridoxal phosphate complex (Fig. 5). 

This byproduct reacts with 3-phosphohydroxypyruvate to give 3-phosphoserine plus regenerated pyridoxal phosphate. 

The second half takes place by the reverse of the preceding pathway. A second a-ketoacid reacts with the enzyme-pyridoxamine phosphate complex (E-PMP) to yield a second amino acid and regenerate the enzyme-pyridoxal phosphate... 

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. 

Fig. 38.4. Function of pyridoxal phosphate (PLP) in transamination reactions. The order in which the reactions occur is 1 to 4. Pyridoxal phosphate (enzyme-bound) reacts with amino acidj, forming a Schiff base (a carbon-nitrogen double bond). After a shift of the double bond, a-keto acidi is released through hydrolysis of the Schiff base, and pyridoxamine phosphate is produced. Pyridoxamine phosphate then forms a Schiff base with a-keto acidj. After the double bond shifts, amino acid2 is released through hydrolysis of the Schiff base and enzyme-bound pyridoxal phosphate is regenerated. The net result is that the amino group from amino acidj is transferred to amino acid2.

Unspecific inhibition of ribonucleotide reduction is produced by compounds like pyridoxal phosphate, or the sulfonated anthraquinone-triazine dye, Cibacron blue. They interact, like in many enzymes, with nucleotide binding domains where pyridoxal phosphate becomes covalently linked to lysine, or in that the dye occupies the whole nucleotide fold. The latter interaction permits its use in affinity chromatography of ribonucleotide reductases Likewise, EDTA is not a specific, nor a potent inhibitor, it may, for example, act by complexation of the structure-stabilizing Mg " ions in native holoenzymes. However the iron-promoted radical regeneration process appears far more susceptible to interference from EDTA ... 

These reactions involve the activities of transaminases and decarboxylases (see p. 210), and over 50 pyridoxal phosphate-dependent enzymes have been identified. In transamination, pyridoxal phosphate accepts the a-amino group of the amino acid to form pyridoxamine phosphate and a keto acid. The amino group of pyri-doxamine phosphate can be transferred to another keto acid, regenerating pyridoxal phosphate. The vitamin is believed to play a role in the absorption of amino acids from the intestine. 

The specificity requirements for this enzyme have been shown to be a free —COOH group, an unsubstituted —NHa group, a jS-carbon atom capable of oxidative attack, and an unsubstituted indole nitrogen atom. Beerstecher and Edmonds " claimed that pyruvate and indole accelerated the reaction autocatalytically. The authors speculate that indole and pyruvate function by regenerating the coenzyme (pyridoxal phosphate) from its binding with tryptophan or tryptophan analogs. 

Pyridoxal phosphate and pyridoxamine phosphate should not be classified as ooenzymes, but rather as prosthetic groups, because they are not split off during the reaction, are not handed along to another enzyme protein, and are regenerated on the same enzyme protein. 

Pyridoxal phosphate is regenerated from pyridoxamine phosphate in a reaction with a keto acid a-keto glutarate and oxaloacetate are very effective NHj acceptors (lower half of diagram). Evidently glutamate (or aspartate) is produced in the process, and the significance of transamination reactions rests primarily on the fact that nitrogen is passed on from here, i.e. from glutamate or aspartate to the final excretion product, urea (cf. Section 8). 

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. 

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. 

Recall from Section 20.2 that pyridoxal 5 -phosphate reacts with the a amino group of an a-amino acid to form an imine, or Schiff base. When L-dopa reacts with PLP, the resultant imine undergoes decarboxylation, with the pyridinium ion of PLP acting as the electron acceptor. Hydrolysis then gives dopamine and regenerated PLP. The mechanism is shown in Figure 25.7. 

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