Pyridoxal phosphate-dependent reaction decarboxylation

Pyridoxal phosphate is a required coenzyme for many enzyme-catalyzed reactions. Most of these reactions are associated with the metabolism of amino acids, including the decarboxylation reactions involved in the synthesis of the neurotransmitters dopamine and serotonin. In addition, pyridoxal phosphate is required for a key step in the synthesis of porphyrins, including the heme group that is an essential player in the transport of molecular oxygen by hemoglobin. Finally, pyridoxal phosphate-dependent reactions link amino acid metabolism to the citric acid cycle (chapter 16). 

Most people have heard of antihistamines, even if they have little concept of the nature of histamine. Histamine is the decarboxylation product from histidine, and is formed from the amino acid by the action of the enzyme histidine decarboxylase. The mechanism of this pyridoxal phosphate-dependent reaction will be studied in more detail later (see Section 15.7). 

Amino acid decarboxylation takes place by the removal of the a-carboxyl group to give the corresponding amine. Two mechanisms of action have been identified which include a pyridoxal phosphate dependent reaction and a non-pyridoxal phosphate dependent reaction. 

Non-pyridoxal Phosphate Dependent. Figure 2 depicts the postulated mechanism for a non-pyridoxal phosphate catal) zed decarboxylation of histidine to histamine involving a pyruvoyl residue instead of pyridoxal -5 - phosphate (20). Histidine decarboxylases from Lactobacillus 30a and a Micrococcus sp. have been shown to contain a covalently bound pyruvoyl residue on the active site. The pyruvoyl group is covalently bound to the amino group of a phenylalanine residue on the enzyme, and is derived from a serine residue (21) of an inactive proenzyme (22). The pyruvoyl residue acts in a manner similar to pyridoxal phosphate in the decarboxylation reaction. 

ALA synthase is a pyridoxal phosphate-dependent enzyme and promotes Schiff-base formation between its coenzyme and glycine (67 in Fig. 37). Nucleophilicity at C-2 of the glycine could be generated either by decarboxylation or by abstraction of a proton. In the first case 5-aminolaevulinic acid would retain both methylene protons of glycine, in the second, one of the protons would be lost to the medium (Fig. 37). Acylation of the pyridoxal-bound intermediate (68 or 69) by succinyl-CoA would constitute the next step and this could be followed either by direct hydrolysis of the Schiff-base or by decarboxylation with subsequent hydrolysis depending on which course was chosen in the first stage of the reaction. 

The same scaffold was used to design catalysts for pyridoxal phosphate-dependent deamination of aspartic acid to form oxaloacetate, one half of the transamination reaction [8], and oxaloacetate decarboxylation [14]. Catalysis was due to binding of pyridoxal phosphate in close proximity to His residues capable of rate limiting 1,3 proton transfer. A two-residue catalytic site containing one Arg and one Lys residue was found to be the most efficient decarboxylation agent, more efficient per residue than the Benner catalyst, most likely due to a combination of efficient imine formation, pK depression and transition state stabilization. 

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. 

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. ... 

Notably, nitrile-degrading enzymes (e.g. nitrilase that converts the CN group to carboxylic acid, and nitrile hydratase that produces an amide function) have been described, and they co-exist with aldoxime-degrading enzymes in bacteria (Reference 111 and references cited therein). Smdies in this area led to the proposal that the aldoxime-nitrile pathway, which is implemented in synthesis of drugs and fine chemicals, occurs as a natural enzymic pathway. It is of interest that the enzyme responsible for bacterial conversion of Af-hydroxy-L-phenylalanine to phenacetylaldoxime, an oxidative decarboxylation reaction, lacks heme or flavin groups which are found in plant or human enzymes that catalyze the same reaction. Its dependency on pyridoxal phosphate raised the possibility that similar systems may also be present in plants . 

E. coli (107, 125). The complexes have recently been reviewed (126). It is possible that lipoamide dehydrogenase also functions in the complexes that oxidatively decarboxylate the a-keto acids resulting from the transamination of valine, isoleucine, and leucine but these have proved difficult to resolve (127). Lipoamide dehydrogenase also functions in the pyridoxal phosphate and tetrahydrofolate-dependent oxidative decarboxylation of glycine in the anaerobic bacterium Peptococcus glyci-nophilus. The reaction in which the protein-bound lipoic acid is reduced is very complex and not yet fully understood the ultimate electron acceptor is NAD+ (112,113,128). 

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 conversion of tyrosine to 3,4-dihydroxyphenylalanine occurs both in vivo in man (590) and in vitro by the action of tissue tyrosinase (205, 688). Mammals can decarboxylate both tyrosine (402,407) and dihydroxyphenyl-alanine (406), tyrosine decarboxylase and dihydroxyphenylalanine (dopa) decarboxylases being quite distinct and separable (405), though both are dependent on pyridoxal phosphate (73, 758, and review 72). In mammals dihydroxyphenylalanine is the most readily decarboxylated of all amino acids, and it is therefore not unreasonable to assume that this is the substrate normally decarboxylated in adrenaline biosynthesis cf. 74, 75). Support for this concept derives from the fact that both the substrate and the product of the reaction (3,4-dihydroxyphenylethylamine diagram 11) can or do occur in the adrenal (298, 299, 802), and the amine is moreover, like adrenaline and noradrenaline, a normal urinary excretion product (245, 404). 

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 5 -phosphate dependent enzymes constitute an important class of proteins involved predominately in amino acid metabolism. The PLP-cofactor is capable of catalyzing a variety of reactions at the a-, [3-, and/or y-carbons of amino acid substrates. These reactions include tranamination, racemization, decarboxylation, and aldoyltic cleavage reactions at the a-carbon and elimina-tion/substitution reactions at either the 3-, or y-position of the amino acid substrate (67-74) The chemical properties of the cofactor (67-71) are responsible for the great diversity of reactions catalyzed by PLP, while reaction specificity is ultimately determined by the active site environment imposed by the surrounding apo-protein to which the cofactor is covalendy bound (69). 

The pyridoxal phosphate (PLP)-dependent enzymes catalyze a diverse set of chemical transformations of amino acids. These include transamination, decarboxylation, and racemization reactions [Eqs. (41-43)],... 

Amino acid metabolism requires the participation of three important cofactors. Pyridoxal phosphate is the quintessential coenzyme of amino acid metabolism (see Chapter 38). All amino acid reactions requiring pyridoxal phosphate occur with the amino group of the amino acid covalently bound to the aldehyde carbon of the coenzyme (Fig. 39.3). The pyridoxal phosphate then pulls electrons away from the bonds around the a-carbon. The result is transamination, deamination, decarboxylation, P-elimination, racemization, and -elimination, depending on which enzyme and amino acid are involved. 

Condensation of L-alanine with a heptaketide CoA thioester and concomitant decarboxylation gave compound 31, which was formed with overall retention of the absolute confguration. The same behavior was verified in reactions catalyzed by enzymes of the a-oxoamine synthetase family which depend on pyridoxal phosphate (PLP) as the cofactor. Ring closure, reduction of carbonyl at position 3, and subsequent elimination of water lead to 2//-azepine 33. The latest steps of the biosynthesis involve the modification of the side chain, leading to the carbonyl group of 30 [21],... 

Non-enzymic Decarboxylation of Amino Acids Pyridoxal phosphate is a co-enzyme for numerous enzymes, notably amino acid decarboxylases, amino acid transaminases, histaminase and probably diamine oxidase - -. 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. 

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. 

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