Pyridoxal phosphate enzymes reaction types

A variety of interesting chemical rearrangements occur in the catabolic pathways of amino acids. It is useful to begin our study of these pathways by noting the classes of reactions that recur and introducing their enzyme cofactors. We have already considered one important class transamination reactions requiring pyridoxal phosphate. Another common type of reaction in amino acid catabolism is one-carbon transfers, which usually involve one of three cofactors biotin, tetrahydrofolate, or A-adenosylmethionine (Fig. 18-16). These cofactors transfer one-carbon groups in different oxidation states biotin transfers carbon in its most oxidized state, CO2... 

The reactions of the Cu(II), Fe(III), and Al(III) chelates of Schiff bases formed by the condensation of pyridoxal with amino acids and peptides were found by Snell to have catalytic properties similar to those of the pyridoxal phosphate enzymes. A typical metal-catalyzed reaction of this type would be the transamination of pyridoxal and alanine according to... 

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. 

The metabolism of P-hydroxy-a-amino adds involves pyridoxal phosphate-dependent enzymes, dassified as serine hydroxymethyltransferase (SHMT) (EC 2.1.2.1) or threonine aldolases (ThrA L-threonine selective = EC 4.1.2.5, L-aHo-threonine selective = EC 4.1.2.6). Both enzymes catalyze reversible aldol-type deavage reactions yielding glycine (120) and an aldehyde (Eigure 10.45) [192]. 

Biotin (5) is the coenzyme of the carboxylases. Like pyridoxal phosphate, it has an amide-type bond via the carboxyl group with a lysine residue of the carboxylase. This bond is catalyzed by a specific enzyme. Using ATP, biotin reacts with hydrogen carbonate (HCOa ) to form N-carboxybiotin. From this activated form, carbon dioxide (CO2) is then transferred to other molecules, into which a carboxyl group is introduced in this way. Examples of biotindependent reactions of this type include the formation of oxaloacetic acid from pyruvate (see p. 154) and the synthesis of malonyl-CoA from acetyl-CoA (see p. 162). 

Due to the absence of a hydrogen atom on the a-carbon, the a-fluoroalkyl amino acids (except, of course, the fluoroalanines, vide supra) cannot undergo an elimination of HR Consequently, they are more stable than fluoroalanines and other jS-fluoro amino acids previously described. On the other hand, similar to proteogenic amino acids, jS-fluoro amino acids and a-fluoroalkyl amino acids are generally substrates of pyridoxal phosphate depending on enzymes such as racemases and decarboxylases. When an amino acid is a substrate of such enzymes, the enzyme induces the development of a negative charge on the a-carbon, which can initiate a /(-elimination process. This reaction affords an electrophilic species (Michael acceptor type), which is able to add a nucleophilic residue of the enzyme. This notion of mechanism-based inhibitor is detailed in Chapter 7. 

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-... 

Transamination, often also referred to as aminotransfer, is applied to those enzymatic reactions in which an amino group is exchanged between an amino acid and an a-keto acid. This type of reaction is catalyzed by a group of transferases called transaminases or aminotransferases. They are active in both the cytosol and the mitochondria of most cells. An essential prosthetic group of such enzymes is pyridoxal phosphate, and the reaction is generally of the ping-pong type. 

The coenzyme form of pyridoxine is known as pyridoxal phosphate (PP) The most common type of reaction requiring PP as a coenz5mie is transamination. Enzymes catalysing such reactions are known as transaminases or aminotransferases. The coenzyme binds to its apoenzyme via Schiff s base between its aldehyde group and the epsilon amino group of a lysine in the... 

Another subset of group transfer reactions consists of transaminations (Fig. 8.20). In this type of reaction, the nitrogen group from an amino acid is donated to an a-keto acid, forming a new amino acid and the a-keto acid corresponding to the donor amino acid. Enzymes catalyzing this last type of reaction are called transaminases or aminotransferases. The coenzyme pyridoxal phosphate is reqnired for all transaminases (see Fig. 8.13). 

Figure 11.13 Reactions at a-carbon of a-amino acids catalyzed by pyridoxal enzymes All three substituents at C are subject to labilization in the three types of a-carbon reactions. The hydrogen is labilized in recemization reactions, the amino group is labUized in the transamination and the carboxyl group is labilized in decarboxylation. a-Amino acid condenses with pyridoxal phosphate to yield pyridoxylidene imino acid (an aldimine). The common intermediate, aldimine and distinct ketimines leading to the production of oxo-acid (in transamination), amino acid (in racemization) and amine (in decarboxylation) are shown. The catalytic acid (H-A-) and base (-B ) are symbolic both can be from the same residue such as Lys258 in aspartate aminotransferase.

Although the 20 amino acids and complex folding patterns open up a range of chemical possibilities to proteins, there are also many limitations. In order to widen the range of chemistry available to enzyme catalysts, we make use of auxiliary compounds particularly well suited to certain types of reaction (Fig. A6.1). Thus NAD+, NADP+ and FAD are nucleotide cofactors based on the stmctures of nicotinamide and riboflavin, and are used for oxidation-reduction reactions. Biotin is used for carboxylation, pyridoxal phosphate is used for transamination and decarboxylation and so on. As mentioned in Box 3 (Topic 10), most of these are derived from vitamins in the diet. 

Several types of enzyme involved in amino-acid metabolism (including aminotransferases and decarboxylases) require pyridoxal phosphate (7, PLP) as co-factor. It has been postulated that all such reactions involve the formation of a Schiff-base intermediate between the amino-acid and (7) as the first step. Simple systems containing metal ion, amino-acid, and... 

Vitamin Bg (pyridoxine also known as pyridoxol, 118) is an essential growth factor in the diet of many organisms and animals. It forms part of a coenzyme (pyridoxylphosphate) and it is a cofactor for a class of enzymes known as transaminases. A transaminase or an aminotransferase is an enzyme that catalyzes a type of reaction between an amino acid and an a-keto acid. The presence of elevated transaminase levels can be an indicator of liver damage. Vitamin Bg has both an aldehyde form (pyridoxal, 119) and an amine form (pyridoxamine, 120), and it is known that pyridoxal phosphate is a carrier of amino groups and sometimes amino acids. ... 

Reactions of type (i)-(iv) above result as a consequence of labilization of bond I, aldol-type reactions from labilization of bond 2, cind decarboxylation reactions from labilization of bond 3. Studies of these non-enzymatic reactions, which have been summarized in detail elsewhere. s, provide the principal experimental basis for current views of the mechanism of action of pyridoxal phosphate-dependent enzymes, which catalyze closely simileir reactions in living tissues. ... 

Model reactions of this type have been studied in which the catalyst is pyridoxal plus a metal. The enzymatic reactions all appear to use pyridoxal phosphate as a cofactor, and in the case of a bacterial system, Mn" is also required. A major difference between the enzymatic and the model reactions is the requirement for a folic acid cofactor in the former. The formation of glycine and acetaldehyde from L-threonine and L-allo-threonine has been described by Lin and Greenberg. Their partially purified enzyme, threonine aldolase, was not shown to require any cofactors, and the reaction was not reversed. This is in contrast to the results of nonenzymatic experiments in which pyridoxal and a metal catalyze the reversible cleavage of threonine. 

The pyridoxal phosphate returns to its original form at the end of the reaction. In the case of other coenzymes, a second enzyme reaction must occur before the coenzyme is reconverted to its original form. For example, with NAD (page 214), which is a coenzyme for many dehydrogenase enzymes, the oxidized form of the coenzyme is reduced in the first enzyme reaction to NADH which must then be reoxidized by a second enzyme-catalysed reaction before it is able to participate again in the first type of reaction. 

Pyridoxine, the parent substance of pyridoxal phosphate, is also known as vitamin Be (page 165). Many coenzymes contain a derivative of one or other of the B vitamins as an essential part of their structure. Coenzymes may react with a number of different enzymes which are specific for different substrates but which catalyse the same general type of reaction. 

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). 

The mechanism of this enzymic reaction, catalyzed by pyridoxal phosphate, has been discussed above as pathway b (Section 4). The net result is the production of CO2 and a primary amine, whose formula can easily be derived from the amino acid which was decarboxylated. Amines of this type are called biogenic amines (Guggenheim) many of them possess a strong pharmacologic effect, and others are important as precursors of hormones and as components of coenzymes and other active substances. 

Applications of tryptophan synthetase Tryptophan synthetase (EC 4.2.1.20) is a pyridoxal phosphate-dependent enzyme that, in the cell, catalyzes the a,/3-elimination of water from serine to form a pyridoxyl-bound a-aminoacrylate, which undergoes Michael addition of indole to form the named amino acid. This type of reaction has been used to prepare (5)-tryptophan isotopomers with a variety of labeling patterns by use of different labeled indoles and (5)-serines in yields of up to 98% based on indole and 92% based on (5)-serine. 

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). 

ACS isozyme utilizes pyridoxal-5 -phosphate (PLP) as a cofactor and belongs to fold type I PLP-dependent enzymes showing an absorption maximum between 422 and 431 nm, which is due to the internal aldimine. The reaction mechanism proposed for the conversion of SAM to ACC by ACS illustrated in Scheme 2 involves the following steps ... 

Recently, we have modeled9 intrinsic carbon kinetic isotope effects on the ornithine decarboxylase-catalyzed decarboxylations. Decarboxylations occur from the pyridoxal 5 -phosphate (PLP) - substrate complexes. These reactions provide a good model case since a number of 13C kinetic isotope effects for the wild-type enzyme and its mutants, as well as for physiological and slow substrates, have been reported.10 Using AM1/CHARMM/MD calculations on nearly 18000-atom models... 

The glycine-dependent aldolases are pyridoxal 5-phosphate dependent enzymes that catalyze the reversible aldol reaction, where glycine and an acceptor aldehyde form a (i-hydroxy-a-amino acid (Scheme 5.47).74 Serine hydroxymethyltransferases, SHMT (EC 2.1.2.1), and threonine aldolases, two types of glycine dependent aldolases, have been isolated. In... 

L-Amino acid transaminases are ubiquitous in nature and are involved, be it directly or indirectly, in the biosynthesis of most natural amino acids. All three common types of the enzyme, aspartate, aromatic, and branched chain transaminases require pyridoxal 5 -phosphate as cofactor, covalently bound to the enzyme through the formation of a Schiff base with the e-amino group of a lysine side chain. The reaction mechanism is well understood, with the enzyme shuttling between pyridoxal and pyridoxamine forms [39]. With broad substrate specificity and no requirement for external cofactor regeneration, transaminases have appropriate characteristics to function as commercial biocatalysts. The overall transformation is comprised of the transfer of an amino group from a donor, usually aspartic or glutamic acids, to an a-keto acid (Scheme 15). In most cases, the equilibrium constant is approximately 1. 

The most useful, and thus far successful, examples have involved irreversible reactions of nucleophilic functions of an enzyme s reactive site with an enzymatically activated Kcat inhibitor of a Michael-type addition reaction. The activation invariably requires participation of the enzyme s prosthetic group (e.g., flavin of monoamine oxidase) or coenzymes such as pyridoxal (vitamin B) as its phosphate, which is associated with several enzymes (e.g., threonine dehydrase, ornithine decarboxylase, a-ketoglutarate transaminase). 

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