Pyridoxal function

Chapter V. Amino group transfer (Pyridoxal function) by E. E. Snell (Berkeley, Calif.)... 

Pyridoxal Derivatives. Various aldehydes of pyridoxal (Table 3) react with hemoglobin at sites that can be somewhat controlled by the state of oxygenation (36,59). It is thereby possible to achieve derivatives having a wide range of functional properties. The reaction, shown for PLP in Figure 3, involves first the formation of a Schiff s base between the amino groups of hemoglobin and the aldehyde(s) of the pyridoxal compound, followed by reduction of the Schiff s base with sodium borohydride, to yield a covalendy-linked pyridoxyl derivative in the form of a secondary amine. 

B6 function, including pyridoxin (= PN, alcohol), pyridoxal (= PL, aldehyde), pyridoxamine (= PM, amine), and their 5 -phosphoiylised forms. 

Vitamin Ba (pyridoxine, pyridoxal, pyridoxamine) like nicotinic acid is a pyridine derivative. Its phosphorylated form is the coenzyme in enzymes that decarboxylate amino acids, e.g., tyrosine, arginine, glycine, glutamic acid, and dihydroxyphenylalanine. Vitamin B participates as coenzyme in various transaminations. It also functions in the conversion of tryptophan to nicotinic acid and amide. It is generally concerned with protein metabolism, e.g., the vitamin B8 requirement is increased in rats during increased protein intake. Vitamin B6 is also involved in the formation of unsaturated fatty acids. 

Work in the Imperiali laboratory has also focused on exploring the ability of minimal peptide scaffolds to augment the rate of coenzyme-mediated transaminations [22-25]. To accomplish this, a strategy has been developed in which the core functionality of the coenzyme is incorporated as an integral constituent of an unnatural coenzyme amino acid chimera construct. Thus, non-cova-lent binding of the coenzyme to the peptide or protein scaffold is unnecessary. Both the pyridoxal and pyridoxamine analogs have been synthesized in a form competent for Fmoc-based solid phase peptide synthesis (SPPS) (Fig. 7) [23,24]. 

The binding of pyridoxal 5 -phosphate (vitamin Be) to enzymes has been modelled using homo- and co-polypeptides containing L-lysine as a source of reactive amino groups. This has now been extended to reaction of pyridoxal with polyallylamine, with the polymer acting as a control that cannot provide amido -CO- or -NH- functions to stabilize the Schiff base products, as occurs in enzymes and polypeptides. Rate constants for the formation and hydrolysis of the imines have been measured and interpreted in terms of formation of the carbinolamine (in its neutral or zwitterionic form). 

Numerous examples of modiflcations to the fundamental cyclodextrin structure have appeared in the literature.The aim of much of this work has been to improve the catalytic properties of the cyclodextrins, and thus to develop so-called artificial enzymes. Cyclodextrins themselves have long been known to be capable of catalyzing such reactions as ester hydrolysis by interaction of the guest with the secondary hydroxyl groups around the rim of the cyclodextrin cavity. The replacement, by synthetic methods, of the hydroxyl groups with other functional groups has been shown, however, to improve remarkably the number of reactions capable of catalysis by the cyclodextrins. For example, Breslow and CO workersreported the attachment of the pyridoxamine-pyridoxal coenzyme group to beta cyclodextrin, and thus found a two hundred-fold acceleration of the conversion of indolepyruvic acid into tryptophan. 

The cases of myoglobin and hemoglobin are not rare. Many enzymes are dependent for their function on the presence of a nonprotein group. For example, cytochrome c also contains a prosthetic group similar, but not identical, to heme, as do a number of other proteins. These are known generically as heme proteins. There is a family of enzymes that contain a flavin group, the flavoproteins. Another family contains pyridoxal phosphate, a derivative of vitamin Be. There are a number of other examples. 

Notably, nitrile-degrading enzymes (e.g. nitrilase that converts the CN group to carboxylic acid, and nitrile hydratase that produces an amide function) have been described, and they co-exist with aldoxime-degrading enzymes in bacteria (Reference 111 and references cited therein). Smdies in this area led to the proposal that the aldoxime-nitrile pathway, which is implemented in synthesis of drugs and fine chemicals, occurs as a natural enzymic pathway. It is of interest that the enzyme responsible for bacterial conversion of Af-hydroxy-L-phenylalanine to phenacetylaldoxime, an oxidative decarboxylation reaction, lacks heme or flavin groups which are found in plant or human enzymes that catalyze the same reaction. Its dependency on pyridoxal phosphate raised the possibility that similar systems may also be present in plants . 

Tiburzy (22,31) obtained similar results by application of the PAL inhibitor aminooxyacetic acid (AOA). However, AOA does not specifically inhibit PAL (99), and PAL is not only involved in lignin biosynthesis (100). Thus, AOA and the related inhibitor aminooxyphenyl propionic acid (AOPP) (101,102) inhibit the biosynthesis of lignin (103,104), anthocyanins (105), other flavonoids (106), and conjugates of cinnamic acids (107) via PAL, as well as ethylene (108-110) via a pyridoxal phosphate dependent enzyme (110,111). In view of the possible function of phenolic compounds as phytoalexins (21,112,113) and the well documented role of ethylene in some resistance reactions (114-116), the above cited experiments with AOA (22,... 

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. 

Suicide Enzyme Inhibitors. Snicide substrates are irreversible enzyme inhibitors that bind covalently. The reactive anchoring group is catalytically activated by the enzyme itself through the enzyme-inhibitor complex. The enzyme thus produces its own inhibitor from an originally inactive compound, and is perceived to commit suicide. To design a substrate, the catalytic mechanism of the enzyme as well as the nature of the functional gronps at the enzyme active site must be known. Conversely, successful inhibition provides valuable information about the structure and mechanism of an enzyme. Componnds that form carbanions are especially usefnl in this regard. Pyridoxal phosphate-dependent enzymes form such carbanions readily becanse... 

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). 

Vitamin B6 occurs naturally in three related forms pyridoxine (6.26 the alcohol form), pyridoxal (6.27 aldehyde) and pyridoxamine (6.28 amine). All are structurally related to pyridine. The active co-enzyme form of this vitamin is pyridoxal phosphate (PLP 6.29), which is a co-factor for transaminases which catalyse the transfer of amino groups (6.29). PLP is also important for amino acid decarboxylases and functions in the metabolism of glycogen and the synthesis of sphingolipids in the nervous system. In addition, PLP is involved in the formation of niacin from tryptophan (section 6.3.3) and in the initial synthesis of haem. 

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... 

T Although D-amino acids do not generally occur in proteins, they do serve some special functions in the structure of bacterial cell walls and peptide antibiotics. Bacterial peptidoglycans (see Fig. 20-23) contain both D-alanine and D-glutamate. D-Amino acids arise directly from the l isomers by the action of amino acid racemases, which have pyridoxal phosphate as cofactor (see Fig. 18-6). Amino acid racemization is uniquely important to bacterial metabolism, and enzymes such as... 

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. 

Vitamin B6 (pyridoxine, pyridoxamine, and pyridoxal) has the active form, pyridoxal phosphate. It functions as a cofactor for enzymes, particularly in amino acid metabolism. Deficiency of this vitamin is rare, but causes glossitis and neuropathy. The deficiency can be induced by isoniazid, which causes sensory neuropathy at high doses. 

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... 

Observation of an abnormally large shift in the position of fluorescent emission of pyridoxal phosphate (PLP) in glycogen phosphorylase answered an interesting chemical question.187188 A 330 nm (30,300 cm ) absorption band could be interpreted either as arising from an adduct of some enzyme functional group with the Schiff base of PLP and a lysine side chain (structure A) or as a nonionic tautomer of a Schiff base in a hydrophobic environment (structure B, Eq. 23-24). For structure A, the fluorescent emission would be expected at a position similar to that of pyridoxamine. On the other hand, Schiff bases of the... 

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