Enzymes, decarboxylation decarboxylase

By contrast, the cytoplasmic decarboxylation of dopa to dopamine by the enzyme dopa decarboxylase is about 100 times more rapid (Am 4x 10 " M) than its synthesis and indeed it is difficult to detect endogenous dopa in the CNS. This enzyme, which requires pyridoxal phosphate (vitamin B6) as co-factor, can decarboxylate other amino acids (e.g. tryptophan and tyrosine) and in view of its low substrate specificity is known as a general L-aromatic amino-acid decarboxylase. 

Histamine is synthesised by decarboxylation of histidine, its amino-acid precursor, by the specific enzyme histidine decarboxylase, which like glutaminic acid decarboxylase requires pyridoxal phosphate as co-factor. Histidine is a poor substrate for the L-amino-acid decarboxylase responsible for DA and NA synthesis. The synthesis of histamine in the brain can be increased by the administration of histidine, so its decarboxylase is presumably not saturated normally, but it can be inhibited by a fluoromethylhistidine. No high-affinity neuronal uptake has been demonstrated for histamine although after initial metabolism by histamine A-methyl transferase to 3-methylhistamine, it is deaminated by intraneuronal MAOb to 3-methylimidazole acetic acid (Fig. 13.4). A Ca +-dependent KCl-induced release of histamine has been demonstrated by microdialysis in the rat hypothalamus (Russell et al. 1990) but its overflow in some areas, such as the striatum, is neither increased by KCl nor reduced by tetradotoxin and probably comes from mast cells. 

Kluger and Brandi (1986b) also studied the decarboxylation and base-catalysed elimination reactions of lactylthiamin, the adduct of pyruvate and thiamin (Scheme 2). These reactions are nonenzymic models for reactions of the intermediates formed during the reaction catalysed by the enzyme pyruvate decarboxylase. The secondary j3-deuterium KIE for the decarboxylation was found to be 1.09 at pH 3.8 in 0.5 mol dm-3 sodium acetate at 25°C. In the less polar medium, 38% ethanolic aqueous sodium acetate, chosen to mimic the nonpolar reactive site in the enzyme, the reaction is significantly faster but the KIE was, within experimental error, identical to the KIE found in water. This clearly demonstrates that the stabilization of the transition state by hyperconjugation is unaffected by the change in solvent. 

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). 

An important example of PLP-dependent amino acid decarboxylation is the conversion of histidine into histamine. Histamine is often involved in human allergic responses, e.g. to insect bites or pollens. Stress stimulates the action of the enzyme histidine decarboxylase and histamine is released from mast cells. Topical antihistamine creams are valuable for pain relief, and oral antihistamines are widely prescribed for nasal allergies such as hay fever. Major effects of histamine include dilation of blood vessels, inflammation and swelling of tissues, and narrowing of airways. In serious cases, life-threatening anaphylactic shock may occur, caused by a dramatic fall in blood pressure. 

The principal pathways for the biogenesis and metabolism of histamine are well known. Histamine is formed by decarboxylation of the amino acid, L-histidine, a reaction catalyzed by the enzyme, histidine decarboxylase. This decarboxylase is found in both mammalian and non-mammalian species. The mammalian enzyme requires pyridoxal phosphate as a cofactor. The bacterial enzyme has a different pH optimum and utilizes pyruvate as a cofactor (26.27). 

The lyases comprise enzyme class 4. They are enzymes cleaving C-C, C-0, C-N and other bonds by elimination, not by hydrolysis or oxidation. Lyases also catalyse addition to donble bonds. The types of reactions catalysed by lyases are decarboxylation (decarboxylase), hydration/dehydration (hydratase/dehydratase), ammonia addition/deamination (ammonia-lyase), cyanohydrin formation/cleavage (oxynitrilase),... 

Histamine is an imidazole compound, formed by decarboxylation of the amino acid L-histidine, a reaction catalyzed by the enzyme histidine decarboxylase. 

Histamine is formed by decarboxylation of the amino acid l -histidine, a reaction catalyzed in mammalian tissues by the enzyme histidine decarboxylase. Once formed, histamine is either stored or rapidly inactivated. Very little histamine is excreted unchanged. The major metabolic pathways involve conversion to /V-methylhistamine, methylimidazoleacetic acid, and imidazoleacetic acid (IAA). Certain neoplasms (systemic mastocytosis, urticaria pigmentosa, gastric carcinoid, and occasionally myelogenous leukemia) are associated with increased numbers of mast cells or basophils and with increased excretion of histamine and its metabolites. 

Specific decarboxylases for most of the common amino acids have been isolated. In mammals, a decarboxylase involved in the biosynthesis of neuroactive amines is particularly important. This enzyme decarboxylates 3,4-dihydroxyphenylalanine and 5-hydroxytryp-tophan (both products of tetrahydrobiopterin-dependent hydroxylations—Section 1.10.5.1) to give 3,4-dihydroxyphenethylamine and serotonin (equation 10), respectively (70MI11002). 

There are significant differences in the reactivity of synthetic intermediate analogues for these reactions and the corresponding intermediates in the enzymic system. Lienhard and coworkers42,43 reported that the rate of decarboxylation of 2-(l-carboxy-l-hydroxyethyl)-3,4-dimethylthiazolium chloride is very fast relative to pyruvate (whose reaction is too slow to observe) but slower than the enzymic decarboxylation of pyruvate decarboxylase (PDC) by a factor of 105. Similar observations of a catalytic gap were seen for the rate of decarboxylation of lactylthiamin compared to PDC and the... 

Although the utility of transaminases has been widely examined, one such limitation is the fact that the equilibrium constant for the reaction is near unity. Therefore, a shift in this equilibrium is necessary for the reaction to be synthetically useful. A number of approaches to shift the equilibrium can be found in the literature.53 124135 Another method to shift the equilibrium is a modification of that previously described. Aspartate, when used as the amino donor, is converted into oxaloacetate (32) (Scheme 19.21). Because 32 is unstable, it decomposes to pyruvate (33) and thus favors product formation. However, because pyruvate is itself an a-keto acid, it must be removed, or it will serve as a substrate and be transaminated into alanine, which could potentially cause downstream processing problems. This is accomplished by including the alsS gene encoding for the enzyme acetolactate synthase (E.C. 4.1.3.18), which condenses two moles of pyruvate to form (S)-aceto-lactate (34). The (S)-acetolactate undergoes decarboxylation either spontaneously or by the enzyme acetolactate decarboxylase (E.C. 4.1.1.5) to the final by-product, UU-acetoin (35), which is meta-bolically inert. This process, for example, can be used for the production of both l- and d-2-aminobutyrate (36 and 37, respectively) (Scheme 19.21).8132 136 137... 

Another example where mechanism and model have been developed is that for the decarboxylation of acetoacetic acid here no coenzyme is required, and the chemistry involves the enzyme itself. The mechanism for the enzymic decarboxylation with crystalline decarboxylase from Clostridium acetobutylicum has been worked out in some detail it is presented below (20, 21). The initial work, carried out in the author s laboratory by G. Hamilton (22) and I. Fridovich (23, 24) proved that the essential intermediate is a ketimine much of the subsequent development of the enzymic system resulted from the researches of W. Tagaki (25). 

AGM (decarboxylated arginine), an endogenous amine derived from arginine and its biosynthetic enzyme (arginine decarboxylase), is broadly distributed in the CNS, including the SDH (Li et al., 1994a Raasch et al., 1995 Reis and Regunathan,... 

To solve this problem, we used a mimic of a different enzyme, diaUcylglycine decarboxylase (30). In this enzyme, pyridoxal phosphate reacts with an alpha-disubstituted glycine to perform an irreversible decarboxylation (Fig. 6) while converting the pyridoxal species to a pyridoxamine. We imitated this with our model transaminations using pyridoxal species that carry hydrophobic chains, and we were able to achieve as many as 100 catalytic turnovers. Thus, we could imitate one enzyme—the ordinary transaminases—by also imitating another enzyme that solved the turnover problem. 

Can we apply any of this information from non-enzymatic catalysis to decarboxylating enzymes Some decarboxylases do form Schiff bases with their substrates, and some are dependent on metal ions. The acetone-forming fermentation of Clostridium acetobutylicum requires large amounts of acetoacetate decarboxylase (Eq. 13-44). 

Another example is to be found in the drug therapy of Parkinson s disease. The use of L-dopa (levodopa) as a prodrug for dopamine has already been described. However, to be effective, large doses of L-dopa (3-8 g per day) are required, and over a period of time these dose levels lead to side-effects such as nausea and vomiting. L-Dopa is susceptible to the enzyme dopa decarboxylase and as a result, much of the L-dopa administered is decarboxylated to L-dopamine before it reaches the central nervous system (Fig. 8.21). 

A very important naturally occuring thiazole derivative is thiamine pyrophosphate (473). It is the prosthetic group in a variety of enzymes which catalyze decarboxylation (decarboxylase) and aldol-type condensation (aldolase) reactions. The catalytic active site of the molecule is at C-2 of the thiazole ring . The same activity of (473) is shown by other thiazolium salts and therefore these compounds have been widely exploited as catalysts in reactions of importance such as the benzoin condensation (see Section 3.06.12.2). 

The decarboxylation reaction catalyzed by the enzyme oxaloacetate decarboxylase has been examined using enzymes from four different sources Pseudomonas putida, Micrococcus luteus, and two strains of Azotobacter vinelandii. The highest rates were obtained with the oxaloacetate decarboxylase isolated from Pseudomonas, a Mg2+-requiring enzyme17, 121. 

The mechanism of the enzymic decarboxylation of oxaloacetate presumably resembles that of the metal ion-catalyzed reaction (Scheme III), in which the enzyme-bound metal chelates to the a-carboxyl and the keto carbonyl of the substrate prior to decarboxylation (cf. Scheme II). Interestingly, an enzyme identified as oxaloacetate decarboxylase was later identified as pyruvate kinase... 

Like 6-phosphogluconate dehydrogenase and UDPglucuronate decarboxylase, this enzyme decarboxylates a /3-keto acid with an a-oxygen function without the involvement of a metal ion or other cofactor (70). Apparently the electronic properties of this oxygen function are sufficient to stabilize the enolate anion intermediate, so that no metal ion is needed. 

Histamine, which is widely distributed in nature, is formed by the decarboxylation of L-histidine by the enzyme histidine decarboxylase. Non-enzymic decarboxylation of histidine hais also been observed but the conditions under which this occurs render the reaction of little physiological interest. 

The results show that if the extract contained two individual enzymes, DOPA decarboxylase and 5-HTP decarboxylase, then not only must DOPA have the same affinity for each enzyme, but 5-HTP must also have the same affinity for each enzyme. Alternatively the extract could contain only one decarboxylase capable of decarboxylating both DOPA and 5-HTP. As the second alternative is the more probable one, these results provide strong, though by themselves not conclusive, evidence that in the rabbit kidney extract one enzyme is responsible for the decarboxylation of both substrates. 

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