Pyridoxal enzyme reaction

Figure 11.7 Spectral changes accompanying pyridoxal enzyme reaction...

A few years later, in 1953, the versatility of pyridoxal phosphate was illustrated by Snell and his collaborators who found many of the enzyme reactions in which pyridoxal phosphate is a coenzyme could be catalyzed non-enzymically if the substrates were gently heated with pyridoxal phosphate (or free pydridoxal) in the presence of di- or tri-valent metal ions, including Cu2+, Fe3+, and Al3+. Most transaminases however are not metal proteins and a rather different complex is formed in the presence of the apoprotein. 

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 terminology vitamin Bg covers a number of structurally related compounds, including pyridoxal and pyridoxamine and their 5 -phosphates. Pyridoxal 5 -phosphate (PLP), in particular, acts as a coenzyme for a large number of important enzymic reactions, especially those involved in amino acid metabolism. We shall meet some of these in more detail later, e.g. transamination (see Section 15.6) and amino acid decarboxylation (see Section 15.7), but it is worth noting at this point that the biological role of PLP is absolutely dependent upon imine formation and hydrolysis. Vitamin Bg deficiency may lead to anaemia, weakness, eye, mouth, and nose lesions, and neurological changes. 

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). 

E. Krebs (Fischer et al 1958). They discovered that pyridoxal phosphate is attached to phosphorylase A by a ketimine linkage, and that the C=N bond of this linkage could be irreversibly reduced with sodium borohydride. Pyridoxal phosphate does not participate directly in the enzymic reaction of phosphorylase the significance of the work rests on the fact that the reduction occurs without inactivating the enzyme. In 1961, Horecker and his coworkers reduced a mixture of glucose-6-phosphate-14C and transaldolase... 

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. 

Schiff base formation can have a considerable effect on both the position and degree of activation of the coordinated amino acid. The Schiff bases derived from amino acids and pyridoxal have attracted considerable attention due to the biochemical significance of vitamin B6 and the realization that many of the enzymic reactions involving B6 could be brought about in the absence of enzyme by using pyridoxal and various metal ions.444,445,461 4 2,342... 

The alanine racemization catalyzed by alanine racemase is considered to be initiated by the transaldimination (Fig. 8.5).26) In this step, PLP bound to the active-site lysine residue forms the external Schiff base with a substrate alanine (Fig. 8.5, 1). The following a-proton abstraction produces the resonance-stabilized carbanion intermediates (Fig. 8.5, 2). If the reprotonation occurs on the opposite face of the substrate-PLP complex on which the proton-abstraction proceeds, the antipodal aldimine is formed (Fig. 8.5,3). The subsequent hydrolysis of the aldimine complex gives the isomerized alanine and PLP-form racemase. The random return of hydrogen to the carbanion intermediate is the distinguishing feature that differentiates racemization from reactions catalyzed by other pyridoxal enzymes such as transaminases. Transaminases catalyze the transfer of amino group between amino acid and keto acid, and the reaction is initiated by the transaldimination, followed by the a-proton abstraction from the substrate-PLP aldimine to form a resonance-stabilized carbanion. This step is common to racemases and transaminases. However, in the transamination the abstracted proton is then tranferred to C4 carbon of PLP in a highly stereospecific manner The re-protonation occurs on the same face of the PLP-substrate aldimine on which the a-proton is abstracted. With only a few exceptions,27,28) each step of pyridoxal enzymes-catalyzed reaction proceeds on only one side of the planar PLP-substrate complex. However, in the amino acid racemase... 

Pyridoxal Phosphate Reaction Mechanisms Threonine can be broken down by the enzyme threonine dehydratase, which catalyzes the conversion of threonine to a-ketobutyrate and ammonia. The enzyme uses PLP as a cofactor. Suggest a mechanism for this reaction, based on the mechanisms in Figure 18-6. Note that this reaction includes an elimination at the j8 carbon of threonine. 

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 stereochemistry of pyridoxal phosphate-catalyzed reactions was last summarized comprehensively in 1971 by Dunathan [2], who outlined many of the basic concepts in this field. Aspects of PLP catalysis have been discussed in other reviews on enzyme reaction stereochemistry (e.g., [9]), and a brief review, emphasizing their own work, has recently been published by the present authors [ 10]. Much work has been done in this field during the past ten years, most of it supporting the concepts laid out in Dunathan s review, often refining the picture and sometimes modifying the original ideas. 

Table 9.1 Pyridoxal Phosphate-Catalyzed Enzyme Reactions of Amino Acids ...

The ring nitrogen of pyridoxal phosphate exerts a strong electron withdrawing effect on the aldimine, and this leads to weakening of all three bonds about the a-carbon of the substrate. In nonenzymic reactions, all the possible pyridoxal-catalyzed reactions are observed - a-decarboxylation, aminotrans-fer, racemization and side-chain elimination, and replacement reactions. By contrast, enzymes show specificity for the reaction pathway followed which bond is cleaved will depend on the orientation of the Schiff base relative to reactive groups of the catalytic site. As discussed in Section 9.3.1.5, reaction specificity is not complete, and a number of decarboxylases also undergo transamination. 

The -amino groups of lysyl residues serve as attachment sites of a number of coenzymes in proteins (e.g. biotin in pyruvate carboxylase, pyridoxal phosphate in phosphorylase, lipoic acid in lipoate acetyl-transferase) and form covalent intermediates in several enzymic reactions (e.g. transaldolase, aldolase, etc.). Discussion of all of these naturally-occuring derivatives of lysine will not be attempted in this treatise, but the investigator using chemical modification of proteins should be aware of their possible presence and effect on the results of his experiments. It should be noted that e-N-phospholysine has been reported in nucleoside diphosphate kinase (Walinder 1968). 

The vitamin Bg group comprises three natural forms pyridoxine (pyridoxol) (PA/), pyridoxamine (PM), and pyridoxal (PL), which are 4-substituted 2-methyl-3-hydroxyl-5-hydroxymethyl pyridines (Figure 30-13). During metabolic conversions, each vitamer becomes phosphorylated at the 5-hydroxymethyl substituent. Although both pyridox-amine-5 -phosphate (PMP) and pyridoxal-S -phosphate (PLP, P-5 -P) interconvert as coenzyme forms during aminotransferase (transaminase)-catalyzed reactions, PLP is the coenzyme form that participates in the large number of Bg-dependent enzyme reactions. 

Pyridoxal phosphate is the coenzyme in a large number of amino acid reactions. At this point it is convenient to consider together 1,he mechanism of those pyridoxal-dependent reactions concerned with aromatic amino acids. The reactions concerned are (1) keto acid formation (e.g., from kynurenine, above), 2) decarboxylation (e.g., of 5-hydroxytrypto-phan to 5-hydroxytryptamine, p. 106), (3) scission of the side claain (e.g., 3-tyrosinase, p. 78 tryptophanase, p. 110 and kynureninase, above), and 4) synthesis (e.g., of tryptophan from indole and serine, p. 40). Many workers have considered the mechanism of one or more of these reactions (e.g., 24, 216, 361, 595), but a unified theory is primarily due to Snell and his colleagues (summarized in 593). Snell s experiments have been carried out largely in vitro, and it should be emphasized that in vivo it is the enzyme protein which probably directs the electromeric changes. 

Pyridoxal phosphate is a co-enzyme for numerous enzymes, notably amino acid decarboxylases, amino acid transaminases, histaminase and probably diamine oxidase Ais.iw. As most of the evidence on which the mechanism of action of pyridoxal-dependent enzymes is based has been obtained from studies of the non-enzymic interaction of pyridoxal with amino acids, these non-enzymic reactions will be considered first in some detail. 

A detailed theoretical analysis of non-enzymic pyridoxal-catalysed reactions has been carried out by the molecular orbital method . An interesting result of this analysis was the emphasis placed on the increased resonance energy of structures such as XIV) compared with that of the original Schiff base XIII). It was suggested that this increase in resonance energy is the main reason for the labilization of the bonds a), b) or (c), after Schiff base formation. 

The probable mechanism of the enzymic decarboxylation of histidine can, at present, only be inferred from studies of the non-enzymic reactions discussed in the previous section, and from what is known of the mechanism of action of other pyridoxal phosphate-dependent enzymes. 

The enzyme shows a high substrate specificity for AdoMet, and affinity to the substrate is also high with a Km ranging from 12 to 60 pM, and the pH optimum is between 8.5 and 9.5. Interestingly, S-adenosylethionine shows some activity as a substrate. The enzyme reaction is competitively inhibited by AVG and AOA, which are inhibitors of pyridoxal phosphate-linked enzymes. 

In the light of this background we consider an enormous body of experimental work on pyridoxal-P-dependent enzymic reactions which has accumulated since the enunciation of the Braunstein-Snell hypothesis. Broadly speaking, at a crucial stage in these reactions, the cleavage of either the C -COOH or C -H bond is involved. In Sections 2-5 transformations belonging to these two classes are discussed sequentially with major emphasis on the description of events which occur on the... 

Pyridoxal phosphate-dependent enzymic reactions involving C -H bond cleavage... 

Depending upon the relative rates of the reactions participating in the interconversion of various species in the ternary complex, one may expect to see spectropho-tometrically the presence of the quinonoid intermediate of type 6 (Fig. 42) in a number of pyridoxal-P-dependent enzymic reactions such an intermediate would be expected to possess a bathochromically shifted long wavelength absorption maximum. This has indeed been clearly viewed for the ternary complex of serine hydroxymethyltransferase with glycine as shown in Fig. 46, the absorption at 495 nm being attributed to the quinonoid intermediate [94]. 

In this review, we shall concentrate on the stereochemistry of enzymic reactions of amino acids, many of which involve transformations at prochiral centers. We shall use the nomenclature of Hanson (8) to specify the stereochemistry of prochiral atoms and groups as pro-R (Hjj) and pro-S (Hj) and of prochiral faces as Re and Si and the nomenclature of Mislow and Raban (2) to describe prochiral groups as having enantiotopic or diastereotopic relationships. Reviews on the stereochemistry of enzymic reactions of amino acids were published in 1978 (9,10), and since the seminal review by Dunathan in 1971 (11), several reviews comparing the stereochemistry of pyridoxal phosphate-catalyzed enzymic reactions have appeared (12-15). 

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