Pyridoxal-5 -phosphate coenzyme function

Vitamin Bg is a mixture of six interrelated forms pyridoxine (or pyridoxol) (Figure 19.23), pyri-doxal, pyridoxamine, and their 5 -phosphates derivatives. Interconversion is possible between all forms. The active form of the vitamin is pyridoxal phosphate, which is a coenzyme correlated with the function of more than 60 enzymes involved in transamination, deamination, decarboxylation, or desulfuration reactions. 

The first examples of mechanism must be divided into two principal classes the chemistry of enzymes that require coenzymes, and that of enzymes without cofactors. The first class includes the enzymes of amino-acid metabolism that use pyridoxal phosphate, the oxidation-reduction enzymes that require nicotinamide adenine dinucleotides for activity, and enzymes that require thiamin or biotin. The second class includes the serine esterases and peptidases, some enzymes of sugar metabolism, enzymes that function by way of enamines as intermediates, and ribonuclease. An understanding of the mechanisms for all of these was well underway, although not completed, before 1963. 

At the same time, Snell and coworkers used model systems to achieve most of the reactions of the pyridoxal enzymes (Metzler and Snell, 1952a,b Olivard et al., 1952 Ikawa and Snell, 1954a,b Metzler et al 1954a,b Longnecker and Snell, 1957). They too developed the modern mechanisms for the series of reactions and demonstrated the role of the coenzyme as an electron sink by substituting alternative catalysts for pyridoxal phosphate. In particular, they showed that 2-hydroxy-4-nitrobenzaldehyde (Ikawa and Snell, 1954) functioned in their model systems just as did the vitamin its electronic structure is really quite similar (3). 

All aminotransferases have the same prosthetic group and the same reaction mechanism. The prosthetic group is pyridoxal phosphate (PLP), the coenzyme form of pyridoxine, or vitamin B6. We encountered pyridoxal phosphate in Chapter 15, as a coenzyme in the glycogen phosphorylase reaction, but its role in that reaction is not representative of its usual coenzyme function. Its primary role in cells is in the metabolism of molecules with amino groups. 

FIGURE 18-5 Pyridoxal phosphate, the prosthetic group of aminotransferases. (a) Pyridoxal phosphate (PLP) and its aminated form, pyri-doxamine 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... 

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. 

Other enzymes in the aconitase family include isopropylmalate isomerase and homoaconitase enzymes functioning in the chain elongation pathways to leucine and lysine, both of which are pictured in Fig. 17-18.90 There are also iron-sulfur dehydratases, some of which may function by a mechanism similar to that of aconitase. Among these are the two fumarate hydratases, fumarases A and B, which are formed in place of fumarase C by cells of E. coli growing anaerobically.9192 Also related may be bacterial L-serine and L-threonine dehydratases. These function without the coenzyme pyridoxal phosphate (Chapter 14) but contain iron-sulfur centers.93-95 A lactyl-CoA... 

A number of other racemases and epimerases may function by similar mechanisms. While some amino acid racemases depend upon pyridoxal phosphate (Chapter 14), several others function without this coenzyme. These include racemases for aspartate,113 glutamate,114-1153 proline, phenylalanine,116 and diamino-pimelate epimerase.117 Some spiders are able to interconvert d and l forms of amino acid residues in intact polypeptide chains.118119... 

Katunuma and coworkers (1971) described a protease in the rat that hydrolyzes the apoenzymes of a number of pyridoxal phosphate-dependent enzymes it has no effect on other proteins or the holoenzymes. Presumably, it attacks the conserved amino acid sequence around the active lysine residue to which the internal Schiff base is formed. The activity ofthe enzyme is increased some 10- to 20-fold in vitamin Be deficiency, suggesting that its function is to degrade those enzymes that lose their coenzyme more readily, and so make more pyridoxal phosphate available for use by other enzymes. There is also evidence that some pyridoxal phosphate-dependent apoenzymes are modified to become incapable of activation by pyridoxal phosphate, although retaining immunological cross-reactivity with the normal form of the enzyme in vitamin Be deficiency (Nagata and Okada, 1985). 

Aminotransferases utilize a coenzyme - pyridoxal phosphate - which is derived from vitamin B6. The functional part of pyridoxal phosphate (see here) is an aldehyde functional group attached to a pyridine ring. Catalysis involves a Schiff base intermediate (see here). 

Coenzyme A (CoA), biotin, and pyridoxal phosphate are also activation-transfer coenzymes synthesized from vitamins. CoA (CoASH), which is synthesized from the vitamin pantothenate, contains an adenosine 3, 5 -bisphosphate which binds reversibly, but tightly, to a site on an enzyme (Fig. 8.12A). Its functional group, a sulfhydryl group at the other end of the molecule, is a nucleophile that always... 

Pyridoxal phosphate is a cofactor required for several reactions involving amino acid interconversion. Its requirement is increased in relation to the amount of protein in the diet. Some of the deficiency symptoms can be readily correlated with their coenzyme function. Thiamin pyrophosphate is a cofactor for pyruvate dehydrogenase, the activity of which is decreased in the brain as a result of deficiency. Pantothenic acid is required not only for... 

Spectroscopic studies have shown that the chromophoric system of the coenzyme absorbs at 390 nm, in a region clearly separated from the 280 nm peak of the protein moiety. This feature has been exploited for a variety of structural and kinetic studies (31,34,90]. Pyridoxal phosphate, whether in its free form or when bound in the binary or ternary complexes, can exist, depending upon the pH, in a number of ionised states dictated by the pAT values for the various ionisable groups within the molecule. Metzler and coworkers [91] have ascertained spectrophotometrically the pA gS for 3-hydroxypyridine (pAT 5.1, 8.6) and lV-methyl-3-hydroxypyridine (pAT 4.96) and concluded the pAT, of the 3-hydroxy function to be 5.1 and the pyridinium nitrogen to be 8.6 (Fig. 40). This information allows one to interpret the pAT values of 3.1 and 8.3 obtained by Morozov et al. [92] for pyridoxal, as those corresponding to the 3-hydroxy and N-1 groups respectively (Fig. 41). It was noted [92] that the presence of a 5 -phosphate group on pyridoxal had practically no effect on the shape, half-width, or position of the absorption bands and hence pAf s as noted above. 

By transamination, a whole series of other amino adds becomes accessible. Pyridoxal phosphate functions as a coenzyme, which is primarily bonded to a lysine residue of the enzyme. The attack of glutamic acid leads to the corresponding imine. Tautomerisation (proton transfer) and hydrolysis give pyridox-amine phosphate and a-ketoglutaric acid. Pyridoxamine phosphate then reacts with other a-ketoacids, foUowmg the corresponding mechanism. 

Cystathioninuria, a deficiency of cystathionase, is a much rarer and less clearly defined disorder . While the disease has frequently been associated with mental retardation, this may only refiect the type of individual with which testing most frequently occurs. Patients with normal mental function are also known. Nonetheless, the high levels of cystathionine in brain and the mental defects associated with its faulty metabolism, have led to speculation that this thioether has some special role in nervous function. In tissues from at least one patient, there was evidence that the defect was in pyridoxal phosphate binding by cystathionase and that normal enzyme activity could be achieved at abnormally high levels of coenzyme. This is often quoted as the classical example of a binding or K mutant, but not all patients with the disorder give the same effect. 

Of the three related compounds, the most actively functioning is pyridoxal in the form of the phosphate. Pyridoxal phosphate plays a central role as a coenzyme in the reactions by which a cell transforms nutrient amino acids into mixtures of amino acids and other nitrogenous compounds required for its own metabolism. 

The phosphorylated and non-phosphorylated forms of vitamin Bg have various physical and chemical properties. Vitamin Bs in the form of pyridoxal-5 -phosphate (PLP) and to a lesser extent, pyridoxamine-5 -phosphate (PMP), functions as a coenzyme in over 100 enzymatic reactions. All the forms of vitamin Be possess vitamin activity because they can be converted in vivo to pyridoxal. PN, PM and PL are converted to 5 -phosphate by a single kinase enzyme which in the brain and liver is most active with zinc. PNP and PMP are then converted to PLP by flavin dependent oxidase this is the reason why vitamin B2 deficiency causes a fall in available PLP (Holman 1995). Human cells can synthesize PLP from three vitamers via the Bg salvage pathway but cannot synthesize PLP de novo and must obtain it from dietary sources. 

The crucial role played by vitamin Bg in the nervous system and in neuroendocrinology is based on the fact that various putative neurotransmitters as well as taurine, sphingolipids, and polyamines are synthesized by pyridoxal phosphate (PLP)-dependent enzymes. There are numerous biological effects of vitamin Bg unrelated to the role of PLP as a coenzyme. PLP is an antagonist of both the voltage-mediated and the ATP-mediated calcium transport systems. PLP modulates the activities of steroid hormone receptors and transcription factors. The preventive effect of vitamin Bg on tumorigenesis might also derive from the antioxidant functions of this vitamin. 

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. 

The function of a coenzyme is well illustrated by the role of pyridoxal phosphate in the transfer of amino groups. The enzyme alanine aminotransferase glutamate-pyruvate transaminase) catalyses the reaction of glutamate with pyruvate to form 2-oxoglutarate and alanine. In this reaction, the amino group of glutamate is transferred first to pyridoxal phosphate and then to pyruvate with the formation of alanine. 

Pyridoxal phosphate (PLP) is at the heart of chemistry conducted by a number of enzymes. Many of us know the coenzyme pyridoxal phosphate by the closely related vitamin from which it is derived in our diet—pyridoxine, or vitamin 06. Wheat is a good dietary source of vitamin 06. Although pyridoxal phosphate (see below and the model) is a member of the aldehyde family, when it is involved in biological chemistry, it often contains the closely related functional group with a carbon-nitrogen double bond, the imine group. 

---