Pyridoxal 51-phosphate

Pyridoxal phosphate is the coenzyme for the enzymic processes of transamination, racemization and decarboxylation of amino-acids, and for several other processes, such as the dehydration of serine and the synthesis of tryptophan that involve amino-acids (Braunstein, 1960). Pyridoxal itself is one of the three active forms of vitamin B6 (Rosenberg, 1945), and its biochemistry was established by 1939, in considerable part by the work of A. E. Braunstein and coworkers in Moscow (Braunstein and Kritzmann, 1947a,b,c Konikova et al 1947). Further, the requirement for the coenzyme by many of the enzymes of amino-acid metabolism had been confirmed by 1945. In addition, at that time, E. E. Snell demonstrated a model reaction (1) for transamination between pyridoxal [1] and glutamic acid, work which certainly carried with it the implication of mechanism (Snell, 1945). 

Nevertheless, the full-blown mechanism that showed the role of the coenzyme was only written out in detail by Braunstein and M. M. Shemyakin in 1953 (Braunstein and Shemyakin, 1952, 1953). Their formulae (2), complete with the curved arrow notation of physical organic chemistry, clearly pointed out the role of the coenzyme as an electron sink in a ketimine mechanism. They showed how the coenzyme can function in transamination, racemization and, with some help from Hanke and his collaborators (Mandeles et al 1954), in decarboxylation. The mechanisms they advanced were exactly what we would postulate today, and constituted an early and successful application of theory to mechanistic enzymology. But it must be admitted that the theory appealed because it was reasonable the authors had little or no evidence, in terms of physical organic chemistry, to support their formulation, which is shown in part below. 

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). 

Pyridoxal Phosphate.—Analogues of pyridoxal and pyridoxamine 5 -phosphates have frequently been used to probe the size and shape of the active sites of a number of enzymes. For example, the apoenzyme of a tryptophanase from Bacillus alvei will bind pyridoxal 5 -phosphate as well as the 2-nor, 2 -methyl, 2 -hydroxy, 6-methyl, and A-oxide analogues.27 No analogue that has been modified at C-4 binds to the enzyme, confirming the absolute requirement for Schiff-base formation between the 

Reagents i, L-lactate oxidase ii, NaBH4 iii, MeOH iv, monoamine oxidase v, proteolytic digest 

Giartosio, C. Borri Valtattorni, A. Orlacchio, and C. Turano, Biochem. Biophys. Res. Comm., 1975, 66, 863. 

Pyridoxal phosphate (Formula 2.12) and pyridox-amine (Formula 2.13), derived from it, are designated as vitamin 65 (cf. 6.3.3) and are essential ingredients of food  

Pyridoxal 5 -phosphate (PLP) is the active form of vitamin B6 (pyridoxine (PN) or pyridoxal (PL)) and an essential cofactor for a large number of enzymes that catalyze a multitude of reactions, including decarboxylation, deamination, racemization, and transamination [1], 

The biochemistry of de novo PLP biosynthesis and molecular cloning of genes coding enzymes has been primarily investigated in the gram-negative bacterium Escherichia coli [2-8], Two distinct pathways for PLP de novo biosynthesis have been identified DXP (1-deoxy-o-xylulose 5-phosphate)-dependent and DXP-independent pathways. 

Biosynthesis qf Heterocycles From Isolation to Gene Cluster, First Edition. Patrizia Diana and Girolamo Cirrincione. 

Drug-Coenzyme Interactions Isoniazid and Pyridoxal Phosphate 

The borohydride reduction of Schiff base formed between a protein and pyridoxal phosphate should be carried out at a mildly acidic pH. Although sodium borohydride is more unstable in acidic solution, this disadvantage is offset by the exceptional reactivity of the Schiff base salts which are formed in mildly acidic solution (pH 4.S-6.5) (Schellen-berg 1963). Reductions have been carried out after the protein and pyridoxal-5-phosphate have been incubated at a pH of 7.5 which is then changed to 4.5 or 6.5 with acetic acid (Rippa et al. 1967 Dempsey and Christensen 1962 Piszkiewicz et al. 1970) or after initial incubation at pH 6.0 (Schnackerz and Noltmann 1971). The relative merit of either 

Prior to reduction with sodium borohydride, octyl alcohol can be added to avoid foaming. Then a solution of 0.05-0.06 M sodium borohydride in either water or 0.001 N sodium hydroxide is added in equal aliquots until roughly a 100-fold molar excess has been added. After each addition, the pH of the reaction mixture should be readjusted to the initial acidic pH by the addition of either acetic acid or hydrochloric acid. The temperatures of the reaction mixture have ranged from 4-25°C. The modified protein can then be isolated either by precipitation, dialysis or gel filtration under conditions where the native protein is normally stable. 

Proof that a lysine residue has been modified can be readily obtained because pyridoxyl derivatives of lysine possess characteristic white-blue fluorescence (Ronchi et al. 1969). In addition, they have a distinctive absorption maximum at 325 nm with of 9710 cm (Fisher et al. 1963). Finally, a radiochemical label can be introduced by reducing the pyridoxal-5-phosphate protein complex with tritium-labelled sodium borohydride. The peptide containing the derivatized lysine can therefore be detected either by fluorimetry, spectrophotometry or radiochemical techniques following routine procedures of proteolytic digestion and fractionation. Acid hydrolysis in 6 N HCl for 24 hr of peptides containing pyridoxal-5-phosphate lysine yields pyridoxyl-lysine since phosphate esters are readily hydrolyzed under these conditions. Pyridoxyl-lysine is eluted between lysine and histidine from a 55 cm column of Beckman 50 resin with 0.15 M citrate buffer pH 5.28. 

Authentic pyridoxyl-lysine has been prepared by Schnackerz and Noltmann (1971) by borohydride reduction of a mixture of poly-L-lysine hydrochloride (20 mg) and 0.38 moles of pyridoxal, which had been allowed to stand for 10 min at 0°C in 50 mM sodium phosphate (pH 6.0). Low molecular weight impurities were removed by dialysis against 50 mM sodium acetate buffer (pH 6.0). After dialysis the resulting product can be hydrolyzed in 6 N HCl to yield pyridoxyl-lysine and some lysine. The latter contaminant can be minimized by 

An alternative procedure which can provide analytically pure pyridoxyl-lysine involves the prior synthesis of e-pyridoxyl-N-acetyl lysine (Dempsey and Snell, 1963). A mixture of 1.20 g of a-N-acetyl lysine and 470 mg of potassium hydroxide was dissolved in 20 ml of absolute methanol. Then 1.37 g of pyridoxal is added and the resulting mixture is stirred for 15 min at 25°C and filtered. 50 mg of PtOj is added and the solution is hydrogenated at room temperature at 1 atmosphere for 1 hr. After removal of catalyst by filtration, the pH of the filtrate is reduced to 6.0 (as measured by moist indicator paper) by the addition of methanolic HCl. Concentration of the reaction mixture to roughly one-third of its original volume causes precipitation of KCl which is then removed by filtration. When the apparent pH is decreased to about pH 4.4 by the addition of more methanolic HCl, the product precipitates and it can be collected and washed extensively with methanol. The yellow product, e-pyridoxyl-a-N-acetyl lysine, melts at 174-174.5°C (Dempsey and Christensen, 1962). 

Acetyl-CoA is the real form of activated acetate where 36.9 kJ/mol (8.8 kcal/mol) is liberated upon hydrolysis (see Chapter 2). Reduced lipoic add is reoxidized with an FAD coenzyme present on the complex. 

Pyridoxine or vitamin Bg is an important dietary requirement. The aldehyde form is called pyridoxal and its phosphate ester is implicated in many enzyme catalyzed reactions of amino acids and amines. The reactions are numerous and pyridoxal phosphate (pyridoxal-P) is surely one of nature s most versatile catalysts or coenzymes. The chemistry that will be emphasized here is one of proton transfer. In transamination (equation 7-1), a process of central importance in nitrogen metabolism, it is converted to pyridoxamine. 

In fact, pyridoxal-P coenzyme catalyzes at least seven very different reactions where acid-base chemistry and tautomerism is fully exploited. 

While it may be surprising that the above diverse reactions require the same cofactor, this will be readily understood when it is realized that these reactions have certain common features. All require imine (Schiff base) formation between the aldehyde carbonyl of the cofactor and the amino group of the substrate. The pyridoxal phosphate becomes an electrophilic catalyst or electron sink, as electrons may be delocalized from the amino acid into the ring structure. It is the direction of this delocalization that dictates the reaction type and in model systems more than one reaction pathway is often observed. Thus the enzyme both enhances the rate of reaction and gives direction to that reaction (see page 428). 

In the transamination process, the pyridoxal coenzyme is transferred from the enzyme-imine intermediate to the substrate-imine. The evidence of an imine function comes from reduction by borohydride which does not yield pyridoxine but shows that a covalent bond is formed with a lysine residue of the enzyme. The protonation of the pyridine ring is also essential for catalysis. 

This coenzyme occupies a unique position, in that it is involved in at least four types of reactions which all appear to be quite different. These are the decarboxylation of amino acids, transamination, and the synthesis and cleavage of tryptophan. However, all these reactions, as we shall see, have one thing in common they involve an a-amino acid and are concerned with either the amino group or the carbon atom adjacent to the amino group. 

Pyridoxine (vitamin Be) was first recognized as an essential factor in animal nutrition. Later it was found to have growth-promoting ability for various strains of bacteria, yeast, molds, fungi, and plants. Snell, in a series of studies with bacteria, noted variations in response to certain natural extracts as source of Be among several bacteria. Out of this work came the appreciation that vitamin Be existed in several different forms to which various test organisms responded differently. The vitamin Be complex is now known to consist of the following compounds pyridoxine, pyridoxal, pyridoxamine, and the phosphorylated forms of the two latter substances. 

Since an enzymatic transamination was already known, the similarity between the chemical and the enzymatic reaction led to the suggestion that vitamin Be might be a coenzyme for enzymatic transamination. Schlenk and SnelP tested the hypothesis by measuring the transaminase activity of tissues from vitamin-Be-deficient rats. They found that deficient tissues had a much lower transaminase ability than normal tissues. Subse- 

Pyridoxal phosphate has been established as a coenzyme in two reactions involving tryptophan. An enzyme has been isolated from Neurospora which catalyzes a synthesis of tryptophane from serine and indole. This reaction requires pyridoxal phosphate. An enzyme has been isolated from E. coli which causes the decomposition of tryptophan to pyruvic acid, indole, and ammonia here too pyridoxal phosphate is a necessary cofactor. 

Vitamin Be occurs in a wide variety of biological tissues. The vitamin occurs predominantly in the bound form as the phosphate of pyridoxal or pyridoxamine. In almost all tissues the pyridoxal phosphate is the major form with the exception of liver, where pyridoxamine phosphate appears to be in great excess over the aldehyde form.  

Pyridoxai is also used to move amino groups between amino-acids and, with different enzymes takes part in a number of other reactions involving amino-acids. An example of the steps by which an amino-acid 

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Coenzymes effecting transfer of groups. Examples of this class are adenosine triphosphate (ATP), biotin, coenzyme A and pyridoxal phosphate. 

Coenzymes effecting isomerization. Pyridoxal phosphate also falls into this class,... 

An example of a biologically important aide hyde is pyridoxal phosphate which is the active form of vitamin Bg and a coenzyme for many of the reac tions of a ammo acids In these reactions the ammo acid binds to the coenzyme by reacting with it to form an imine of the kind shown in the equation Re actions then take place at the ammo acid portion of the imine modifying the ammo acid In the last step enzyme catalyzed hydrolysis cleaves the imme to pyridoxal and the modified ammo acid... 

Hydrolases represent a significant class of therapeutic enzymes [Enzyme Commission (EC) 3.1—3.11] (14) (Table 1). Another group of enzymes with pharmacological uses has budt-ia cofactors, eg, in the form of pyridoxal phosphate, flavin nucleotides, or zinc (15). The synthases, and other multisubstrate enzymes that require high energy phosphates, are seldom available for use as dmgs because the required co-substrates are either absent from the extracellular space or are present ia prohibitively low coaceatratioas. 

Fig. 2. Biosynthetic pathway for epinephrine, norepinephrine, and dopamine. The enzymes cataly2ing the reaction are (1) tyrosine hydroxylase (TH), tetrahydrobiopterin and O2 are also involved (2) dopa decarboxylase (DDC) with pyridoxal phosphate (3) dopamine-P-oxidase (DBH) with ascorbate, O2 in the adrenal medulla, brain, and peripheral nerves and (4) phenethanolamine A/-methyltransferase (PNMT) with. Cadenosylmethionine in the adrenal...

Carbonic anhydrase Pyridoxal phosphate (PLP) Amino groups Aspartate aminotransferase... 

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.)... 

Muscle glycogen phosphorylase is a dimer of two identical subunits (842 residues, 97.44 kD). Each subunit contains a pyridoxal phosphate cofactor, covalently linked as a Schiff base to Lys °. Each subunit contains an active site (at the center of the subunit) and an allosteric effector site near the subunit interface (Eigure 15.15). In addition, a regulatory phosphorylation site is located at Ser on each subunit. A glycogen-binding site on each subunit facilitates prior association of glycogen phosphorylase with its substrate and also exerts regulatory control on the enzymatic reaction. 

Pyridoxal phosphate-dependent enzymes (Schiff base)... 

The versatile chemistry of pyridoxal phosphate offers a rich learning experience for the student of mechanistic chemistry. William Jencks, in his classic text. Catalysis in Chemistry and Enzymology, writes ... 

It has been said that God created an organism especially adapted to help the biologist find an answer to every question about the physiology of living systems if this is so it must be concluded that pyridoxal phosphate was created to provide satisfaction and enlightenment to those enzymologists and chemists who enjoy pushing electrons, for no other coenzyme is involved in such a wide variety of reactions, in both enzyme and model systems, which can be reasonably interpreted in terms of the chemical properties of the coenzyme. Most of... 

Write a reasonable mechanism for the 3-ketosphinganine synthase reaction, remembering that it is a pyridoxal phosphate-dependent reaction. 

Pyridoxal phosphate, a close relative of vitamin B6, is involved in a large number of metabolic reactions. TeJl the hybridization, and predict the bond angles for each nonterminal atom. 

The amino acid methionine is biosynthesized by a multistep roule that includes reaction of an inline of pyridoxal phosphate (PLP) to give an unsaturated imine. which then reacts with cysteine. What kinds of reactions are occurring in the two steps ... 

A heterocycle is a cyclic compound that contains atoms of two or more elements in its ring, usually carbon along with nitrogen, oxygen, or sulfur. Heterocyclic amines are particularly common, and many have important biological properties. Pyridoxal phosphate, a coenzyme sildenafil (Viagra),... 

Most amino acids lose their nitrogen atom by a transamination reaction in which the -NH2 group of the amino acid changes places with the keto group of ct-ketoglutarate. The products are a new a-keto acid plus glutamate. The overall process occurs in two parts, is catalyzed by aminotransferase enzymes, and involves participation of the coenzyme pyridoxal phosphate (PLP), a derivative of pyridoxine (vitamin UJ. Different aminotransferases differ in their specificity for amino acids, but the mechanism remains the same. 

The mechanism of the first part of transamination is shown in Figure 29.14. The process begins with reaction between the a-amino acid and pyridoxal phosphate, which is covalently bonded to the aminotransferase by an iminc linkage between the side-chain -NTI2 group of a lysine residue and the PLP aldehyde group. Deprotonation/reprotonation of the PLP-amino acid imine in steps 2 and 3 effects tautomerization of the imine C=N bond, and hydrolysis of the tautomerized imine in step 4 gives an -keto acid plus pyridoxamine... 

Under optimal conditions (pH = 8.0,67 g T1 L-aspartic add, 30°C, 1 1 ratio of enzyme activities) after addition of pyridoxal phosphate, 76 g l 1 L-phenylalanine could be produced within 72 hours (92% conversion). This illustrates how simple biochemical manipulation can increase productivity dramatically. 

Ap4A, diadenosine tetraphosphate BBG, Brilliant blue green BzATP, 2 - 3 -0-(4-benzoyl-benzoyl)-ATP cAMP, cyclic AMP CCPA, chlorocyclopentyl adenosine CPA, cyclopentyl adenosine CTP, cytosine triphosphate DPCPX, 8-cyclopentyl-1,3-dipnopylxanthine IP3, inosine triphosphate lpsl, diinosine penta phosphate a,p-meATP, a,p-methylene ATP p.y-meATP, p.y-meihylene ATP 2-MeSADP, 2-methylthio ADP 2-MeSAMP, 2-methylthio AMP 2-MeSATP, 2-methylthio ATP NECA, 5 -W-ethylcarboxamido adenosine PPADS, pyridoxal-phosphate-6-azophenyl-2, 4 -disulfonic acid PLC, phospholipase C RB2, reactive blue 2 TNP-ATP, 2, 3 -0-(2,4,6-trinitrophenyl) ATP. 

In general, pyridoxamine and pyridoxin are more stable than pyridoxal. All vitamers are relatively heat-stable in acid media, but heat labile in alkaline media. All forms of vitamin B6 are destroyed by UV light in both neutral and alkaline solution. The majority of vitamin B6 in the human body is stored in the form of pyridoxal phosphate in the muscle, bound to glycogen phos-phorylase. 

Pyridoxal phosphate mainly serves as coenzyme in the amino acid metabolism and is covalently bound to its enzyme via a Schiff base. In the enzymatic reaction, the amino group of the substrate and the aldehyde group of PLP form a Schiff base, too. The subsequent reactions can take place at the a-, (3-, or y-carbon of the respective substrate. Common types of reactions are decarboxylations (formation of biogenic amines), transaminations (transfer of the amino nitrogen of one amino acid to the keto analog of another amino acid), and eliminations. 

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]. 

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