Pyridoxal phosphate Schiff bases, reactions

Nucleophilic catalysis is a specific example of covalent catalysis the substrate is transiently modified by formation of a covalent bond with the catalyst to give a reactive intermediate. There are also many examples of electrophilic catalysis by covalent modification. It will be seen later that in the reactions of pyridoxal phosphate, Schiff base formation, and thiamine pyrophosphate, electrons are stabilized by delocalization. 

The biosynthesis of tryptophan occurs by condensation of L-serine with indole, this reaction is catalyzed by tryptophan synthetase. The enzyme is a pyridoxal-phosphate containing enzyme which catalyzes nucleophilic p-substitution reactions of amino acids. The p-hydroxyl group of serine is substituted by indole by the action of the enzyme. The reaction is thought to proceed via a ketimine intermediate (27) which undergoes elimination to give an aminoacrylate-pyridoxal phosphate Schiff base (28). Addition at the P-carbon of indole followed by reversal of the process constitutes the enzymatic synthesis of L-tryptophan. 

It is well over 40 years since Pfeiffer discovered that certain reactions of a-amino acid esters, in particular, ester exchange, racemization and oxygenation, are effected very readily when their Schiff bases with salicylaldehyde are complexed to a transition metal ion (most notably Cu11). The Schiff bases result from a condensation reaction between a reactive carbonyl group and the amino group of the amino acids. Snell and his co-workers43 were also one of the first to point out that similar reactions also occurred if pyridoxal was used instead of salicylaldehyde, and that there is a close analogy with pyridoxal phosphate-promoted enzymic reactions of a-amino acid metabolism. Since then much work has been due on these and other similar systems and their reactivities. 

Explain the role of pyridoxal phosphate in aminotransferase reactions. Be sure to describe the Schiff base and the ketimine that are involved in the mechanism. 

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. 

FIGURE 18.27 Pyridoxal-5-phosphate forms stable Schiff base adducts with amino acids and acts as an effective electron sink to stabilize a variety of reaction intermediates. 

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. 

Another interesting example is SHMT. This enzyme catalyzes decarboxylation of a-amino-a-methylmalonate with the aid of pyridoxal-5 -phosphate (PLP). This is an unique enzyme in that it promotes various types of reactions of a-amino acids. It promotes aldol/retro-aldol type reactions and transamination reaction in addition to decarboxylation reaction. Although the types of apparent reactions are different, the common point of these reactions is the formation of a complex with PLP. In addition, the initial step of each reaction is the decomposition of the Schiff base formed between the substrate and pyridoxal coenzyme (Fig. 7-3). 

The role of Schiff bases formed between pyridoxal phosphate and amino acid residues as intermediate products in many enzymatic reactions is well known and documented. NMR is an excellent tool for studies of the enzymatic processes involving Schiff bases formation. 

Identification of pyridoxal phosphate as coenzyme suggested the aldehyde group on pyridoxine might form an intermediate Schiff s base with the donor amino acid. Pyridoxamine phosphate thus formed would in turn donate its NH2 group to the accepting a-ketonic acid, a scheme proposed by Schlenk and Fisher. 15N-labeling experiments and, later, the detection of the Schiff s base by its absorption in UV, confirmed the overall mechanism. Free pyridoxamine phosphate however does not participate in the reaction as originally proposed. Pyridoxal phosphate is invariably the coenzyme form of pyridoxine. 

The Schiff base can undergo a variety of reactions in addition to transamination, shown in Fig. 6.4 for example, racemization of the amino acid via the a-deprotonated intermediate. Many of these reactions are catalyzed by metal ions and each has its equivalent nonmetallic enzyme reaction, each enzyme containing pyridoxal phosphate as a coenzyme. Many ideas of the mechanism of the action of these enzymes are based on the behavior of the model metal complexes. 

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). 

Reactions of Schiff bases of pyridoxal 5 -phosphate and several therapeutic hydrazine derivatives are described earlier under Mines. 

Pyridoxal phosphate (4) is the most important coenzyme in amino acid metabolism. Its role in transamination reactions is discussed in detail on p. 178. Pyridoxal phosphate is also involved in other reactions involving amino acids, such as decarboxylations and dehydrations. The aldehyde form of pyridoxal phosphate shown here (left) is not generally found in free form. In the absence of substrates, the aldehyde group is covalently bound to the e-amino group of a lysine residue as aldimine ( Schiffs base ). Pyridoxamine phosphate (right) is an intermediate of transamination reactions. It reverts to the aldehyde form by reacting with 2-oxoacids (see p. 178). 

In the absence of substrates, the aldehyde group of pyridoxal phosphate is covalently bound to a lysine residue of the transaminase (1). This type of compound is known as an aldimine or Schiffs base. During the reaction, amino acid 1 (A, la) displaces the lysine residue, and a new aldimine is formed (2). The double bond is then shifted by isomerization. 

Historically, the amine was an aromatic amine but is now generalized to any amine. A Schiff base, also called an aldimine, is formed in the pyridoxal 5-phosphate-dependent aminotransferase reactions. 

The synthesis pathway of quinolizidine alkaloids is based on lysine conversion by enzymatic activity to cadaverine in exactly the same way as in the case of piperidine alkaloids. Certainly, in the relatively rich literature which attempts to explain quinolizidine alkaloid synthesis °, there are different experimental variants of this conversion. According to new experimental data, the conversion is achieved by coenzyme PLP (pyridoxal phosphate) activity, when the lysine is CO2 reduced. From cadeverine, via the activity of the diamine oxidase, Schiff base formation and four minor reactions (Aldol-type reaction, hydrolysis of imine to aldehyde/amine, oxidative reaction and again Schiff base formation), the pathway is divided into two directions. The subway synthesizes (—)-lupinine by two reductive steps, and the main synthesis stream goes via the Schiff base formation and coupling to the compound substrate, from which again the synthetic pathway divides to form (+)-lupanine synthesis and (—)-sparteine synthesis. From (—)-sparteine, the route by conversion to (+)-cytisine synthesis is open (Figure 51). Cytisine is an alkaloid with the pyridone nucleus. 

L-Canaline is an ineffective antimetabolite of L-ornithine since it has little ability to antagonize ornithine-dependent reactions. On the other hand, it forms a covalently bound Schiff-base complex with the pyridoxal phosphate moiety of Bg-containing enzymes. As such it is a potent inhibitor of many decarboxylases and aminotransferases that utilize this vitamin. 

The second part of the reaction requires pyridoxal phosphate (Fig. 22-18). Indole formed in the first part is not released by the enzyme, but instead moves through a channel from the a-subunit active site to the jS-subunit active site, where it condenses with a Schiff base intermediate derived from serine and PLP. Intermediate channeling of this type may be a feature of the entire pathway from chorismate to tryptophan. Enzyme active sites catalyzing different steps (sometimes not sequential steps) of the pathway to tryptophan are found on single polypeptides in some species of fungi and bacte-... 

Fig. 3. (a) Reaction of pyridoxal 5 -phosphate (PLP) with an amino-terminal amino group of hemoglobin (Hb). The reagent is in the form of a Schiffs base with tris(hydroxymethyl)aminomethane [77-86-1] (Tris) buffer, and the reaction is a transamination, (b) The resulting unstable Scbiff s base is reduced with... 

Figure 14-5 Some reactions of Schiff bases of pyridoxal phosphate, (a) Formation of the quinonoid intermediate, (b) elimination of a (3 substituent, and (c) transamination. The quinonoid-carbanionic intermediate can react in four ways (1—4) if enzyme specificity and substrate structure allow.

The principles of the above reactions form the basis of a series of important metabolic interconversions involving the coenzyme pyridoxal phosphate (structure 2.41). This condenses with amino acids to form a Schiff base (structure 2.42). The pyridine ring in the Schiff base acts as an electron sink which very effectively stabilizes a negative charge. 

NMR studies have been carried out on Schiff bases derived from pyridoxal phosphate and amino acids, since they have been proposed as intermediates in many important biological reactions such as transamination, decarboxylation, etc.90 The pK.d values of a series of Schiff bases derived from pyridoxal phosphate and a-amino adds, most of which are fluorinated (Figure 11), have been derived from H and19F titration curves.91 The imine N atom was found to be more basic and more sensitive to the electron-withdrawing effect of fluorine than the pyridine N atom. Pyridoxal and its phosphate derivative are shown in Figure 12a. The Schiff base formation by condensation of both with octopamine (Figure 12b) in water or methanol solution was studied by 13C NMR. The enolimine form is favoured in methanol, while the ketoamine form predominates in water.92... 

Structures of catalytic intermediates in pyridoxal-phosphate-dependent reactions. The initial aldimine intermediate resulting from Schiff s base formation between the coenzyme and the a-amino group of an amino acid (a). This aldimine is converted to the resonance-stabilized... 

Vanadyl and copper(n) ions catalyse the /J-elimination reaction of O-phospho-threonine in the presence of pyridoxal.429 Equilibrium spectroscopic studies of the threonine-metal ion-pyridoxal system have identified a metal-ion complex of the amino-acid-pyridoxal Schiff base. The catalytic effect of the metal is ascribed to its electron-with drawing effecCIt was suggested that the specific catalytic effect of Cu2 + and V02+ arises from their reluctance to co-ordinate the phosphate in an axial position. Other metal ions such as nickel can also form the Schiff base complex but probably stabilize the phosphothreonine system by chelate formation. 

The thermodynamic and kinetic characteristics of an intramolecular transimination reaction observed in solutions containing pyridoxal-5-phosphate and ethylenediamine have been investigated (75JA6530). The open-chain structure Schiff bases and the cyclic tautomers such as 54 are in equilibrium in aqueous solution over the pH range 7.5-14, but these equilibria are rather complex owing to the different states of the ionization (protonation) in both tautomers. The ring-chain equilibrium constant (the sum of all cyclic tautomers versus all open-chain tautomers) varies by less than a factor of 4 over the pH range 7-14. At pH 14, KT = 1.2 at pH 10,... 

There are several types of evidence that the L-serine derivative that activates the a reaction is the Schiff base formed between aminoacrylate and pyridoxal phosphate (ES III in Fig. 7.6). (1) Amino acids including l- or D-tryptophan and glycine that form tetrahedral,... 

Table 6.1 lists the water-soluble vitamins with their structures and coenzyme forms. Certain portions of the coenzymes are especially important in their biological activities, and they are indicated by arrows. For example, in case of coenzyme A, a thiol ester is formed between its -SH residue and the acyl group being transferred. And in the case of pyridoxal phosphate, its carbonyl residue forms a Schiff base with the amino group of the amino acid that is being decarboxylated. Fat-soluble vitamins (Table 6.2) are also transformed into biologically active substances. However, with the possible exception of vitamin K, these do not operate as prosthetic groups or cosubstrates in specific enzyme reactions. 

The aldehyde group of pyridoxal phosphate accepts the amino group from an amino acid by formation of a Schiff base (Chap. 1). In this process the amino acid is converted to a 2-oxoacid, and pyridoxal phosphate is converted to pyridoxamine phosphate. The amino group on pyridoxamine phosphate can now be transferred to another 2-oxoacid, converting it to an amino acid. In this second reaction, the pyridoxamine phosphate is converted back to pyridoxal phosphate. 

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