Schiff base of pyridoxal phosphate

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.

Raetz, C.R., and Auld, D.S. (1972) Schiff bases of pyridoxal phosphate with active center lysines of ribonuclease A. Biochemistry 11,2229-2236. 

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

Ketimine 121,744s. See also Schiff base from pyridoxal phosphate 742 as electron acceptor 746, 747 a-Ketoacid. See 2-Oxoacid Ketoamine 434s Ketodeoxyoctonate. See KDO Ketone(s), acidity of 46 Khorana, H. Gobind 84 Kidney cells, alkaline phosphatase in 645... 

Formation of a Schiff Base with Pyridoxal Phosphate (PLP) ... 

Unlike other pyridoxal phosphate-dependent enzymes, in which it is the carbonyl group that is essential for catalysis, the internal Schiff base between pyridoxal phosphate and lysine in glycogen phosphorylase can be reduced with sodium borohydride without affecting catalytic activity. Thus, while pyridoxal phosphate is essential for phosphorylase activity, it does not act by the same kind of mechanism as in amino acid metabolism. 

The reaction mechanism consists of formation of a Schiff base by pyridoxal phosphate with a reactive amino group of the enzyme entry of glycine and formation of an enzyme-pyridoxal phosphate-glycine-Schiff base complex loss of a proton from the a carbon of glycine with the generation of a carbanion condensation of the carbanion with succinyl-CoA to yield an enzyme-bound intermediate (a-amino-yS-ketoadipic acid) decarboxylation of this intermediate to ALA and liberation of the bound ALA by hydrolysis. ALA synthesis does not occur in mature erythrocytes. 

Cysteine synthesis in bacteria proceeds via a-aminoacrylic acid bound to the enzyme as a Schiff base with pyridoxal phosphate (Cook and Wedding, 1976). Partially purified cysteine synthase from spinach (Schmidt, 1977a) and Chlorella (Schmidt, 1977b) catalyzes an exchange of sulfide into cysteine, consistent with the above mechanism. The exchange of acetate into OAS that would be expected due to formation of the proposed enzyme intermediate was not tested. 

Preexistence of a Schiff base between pyridoxal phosphate and the enzyme may account for the greatly enhanced rates of enzymatic reactions is compared to the rates of the corresponding non-enzymatic reactions. Subsequent reactions of the enzyme with amino acids must involve Schiff base formation via a fast transimination step (see section IV.D). Once the new Schiff base is formed, the e-amino group of the lysine residue that was originally bound to pyridoxal phosphate is free and is in a favorable position to act as a catalyst in subsequent steps of the enzymatic reaction. 

A second mode of reaction of the quinonoid-carban-ionic intermediate is utilized by plants which synthesize an enzyme that acts on the amino acid S -adenosylmethionine to form a cyclic three-membered ring compound aminocy-clopropane carboxylic acid. This is a major plant hormone. In a third type of reaction a proton is added back to the coenzyme itself (see Fig. 14) to form what is called a ketimine (not illustrated). This is a Schiff base of pyridox-amine phosphate (PMP, Fig. 5) with an a-oxoacid and is an essential intermediate compound in the important process of transamination (Fig. 14). This process is utilized by all living organisms both in the synthesis of amino acids and in the breakdown of excesses of amino acids. The human body forms several amino acids via transamination. As shown in Fig. 15, this is a reversible sequence involving a cyclic interconversion of PLP and PMP in reaction steps of the type illustrated in Fig. 14. 

P NMR studies of Schiff base derivatives of pyridoxal phosphate... 

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. 

Binding of pyridoxal phosphate to peptide PP-42 also appears to be selective for lysine 30. As was indicated by NMR spectroscopy and UV/vis experiments, only one of three potential lysine Schiff bases appeared to form. To determine the site or sites of attachment, the aldimine peptide intermediates were reduced, proteolytically cleaved, and the fragments analyzed by mass spectroscopy. This... 

Isoniazid, carbidopa, and hydralazine are hydrazine derivatives with therapeutic uses. They form Schiff bases with pyridoxal 5 -phosphate, and rate constants for their formation and hydrolysis have been measured in aqueous solution pH-rate profiles are reported and compared with that of hydrazine itself. 

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. 

Lactobacillus delbrueckii. In 1953, Rodwell suggested that the histidine decarboxylase of Lactobacillus 30a was not dependent upon pyridoxal phosphate (11). Rodwell based his suggestion upon the fact that the organism lost its ability to decarboxylate ornithine but retained high histidine decarboxylase activity when grown in media deficient in pyridoxine. It was not until 1965 that E. E. Snell and coworkers (12) isolated the enzyme and showed that it was, indeed, free of pyridoxal phosphate. Further advances in characterization of the enzyme were made by Riley and Snell (13) and Recsei and Snell (14) who demonstrated the existence of a pyruvoyl residue and the participation of the pyruvoyl residue in histidine catalysis by forming a Schiff base intermediate in a manner similar to pyridoxal phosphate dependent enzymes. Recent studies by Hackert et al. (15) established the subunit structure of the enzyme which is similar to the subunit structure of a pyruvoyl decarboxylase of a Micrococcus species (16). 

Pyridoxal phosphate exists in an equilibrium between the aldehyde and its covalent hydrate (as in Eq. 13-1). The aldehyde has a yellow color and absorbs at 390 nm (Fig. 14-9), while the hydrate absorbs at nearly the same position as does PMP. The absorption bands of Schiff bases of PLP are shifted even further to longer wavelengths, with N-protonated forms absorbing at 415-430 nm. Forms with an unprotonated C = N group absorb at shorter wavelengths.149 240... 

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... 

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. 

T. Taguchi, M. Sugiura, Y. Hamada, and I. Miwa, In vivo formation of a Schiff base of aminoguanidine with pyridoxal phosphate, Biochem. Pharmacol., 1998, 55, 1667-1671. 

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