Enzyme-bound pyridoxamine 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]. 

Among the NH2 transfer reactions, transaminations (1) are particularly important. They are catalyzed by transaminases, and occur in both catabolic and anabolic amino acid metabolism. During transamination, the amino group of an amino acid (amino acid 1) is transferred to a 2-oxoacid (oxoacid 2). From the amino acid, this produces a 2-oxo-acid (a), while from the original oxoacid, an amino acid is formed (b). The NH2 group is temporarily taken over by enzyme-bound pyridoxal phosphate (PLP see p. 106), which thus becomes pyridoxamine phosphate. 

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. 

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.

The enzyme utilizes pyridoxamine phosphate (PMP) to transaminate the substrate carbonyl group to form 4,5-diaminovalerate plus bound PEP. A second trans-... 

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. 

CO factors, such as NAD, bind only momentarity to the enzyme others, such as vitamin remain bound to the enzyme before, after, and during the event of catalysis. In the caste of vitamin B -iequiring enzymes, the cofactor occurs in the forms pyridoxal phosphate and pyridoxamine phosphate. 

PLP is the cofactor for a large number of enzymes used in the metabolism of amino acids and related compounds. Some of these enzymes are listed in Table 9.3. In the aminotransferases, the cofaefor form shifts between PLP and PME In glutamate-oxaloacetate aminotransferase, for example, glutamate reacts with the enzyme bound cofactor and is converted to ot-ketoglutarate. Its amino group remains bound to the cofactor, which is changed to the pyridoxamine phosphate form ... 

Figure 18 The two haif reactions of the transamination process. The cofactor (Py) shuttles between the pyridoxal phosphate form (Py-CHO), bound to the enzyme as internal aldimine, and the pyridoxamine phosphate (PM P) form (Py-NHz) ...

FIGURE 18-5 Pyridoxal phosphate, the prosthetic group of aminotransferases. (a) Pyridoxal phosphate (PLP) and its aminated form, pyridoxamine phosphate, are the tightly bound coenzymes of aminotransferases. The functional groups are shaded, (b) Pyridoxal phosphate is bound to the enzyme through noncovalent interactions and a Schiff-base linkage to a Lys residue at the active site. The steps in the formation of a Schiff base from a primary amine and a carbonyl group... 

Meat, meat products, offal and egg yolk are rich sources of vitamin Bg (Table 5.8). In foods of animal origin, the main compounds are pyridoxal and pyridoxamine, especially in the form of phosphate esters. For example, in meat the main form is present as pyridoxal 5 -phosphate (about two thirds of this vitamin are prosthetic groups of enzymes) bound to various proteins (such as imine), to a lesser extent as free pyridoxal 5 -phosphate, followed by pyridoxamine 5 -phosphate. In contrast, milk contains only about 10% of vitamin in bound forms. The vitamin content in milk and cheeses is relatively low. 

Similar results were obtained in Yon s laboratory with aspartate amino transferase from pig heart cytosol, which is a dimer with one PLP bound per subunit. A strong anticooperativity for binding of pyridoxamine phosphate (PMP) was reported (Arrio-Dupont, 1972). In the haloenzyme-apoenzyme hybrid an important decrease of reactivity of Cys 190 (which is entirely accessible in apoenzyme and buried in holoenzyme) also indicated strong coupling between protomers in the enzyme molecule (Cournil, 1975 Cournil and Arrio-Dupont, 1975). Dissociation of the apoenzyme by dilution was... 

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. 

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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