Enzymes, decarboxylating

From the practical viewpoint, enzyme-like synthetic catalysts, or syn-zymes, need not be specific for a given reactant structure. In nature enzymes distinguish among closely related molecules and transform only the substrate for which it is specific. Mixtures of molecules may not be involved in the industrial reaction to be catalyzed. Reaction specificity is, of course, a requirement. A synthetic hydrolase should not catalyze other reactions such as decarboxylation. Enzymes bring about rate enhancements of 10 -lO. A synzyme could be of great practical importance with far less efficiency than the natural enzyme if it is cheap and stable. In other words, a near miss in an attempt to mimic enzymes could be a fabulous success. 

Keywoids Asymmetric decarboxylation. Enzyme, Reaction mechanism, a-Arylpropionic acid. 

Therefore, ThDP-dependent enzymes include the potential of both making and breaking of C-C bonds [1]. All enzymes have in common a ThDP-bound active aldehyde intermediate formed either by decarboxylation or by transfer from a suitable donor compound (e.g., from xylulose-5-phosphate by transketolase). Some of the decarboxylating enzymes also catalyze interesting side-reactions where two aldehydes are joined, resulting in so-called acyloin condensations [1,... 

In Leuconostoc oenos ML 34, we have shown oxaloacetic acid decarboxylation manometrically (6, 7, 8). We were also able to demonstate fluorometrically the enzymatic production of reduced NAD with malic acid as a substrate, but, of course, were unable to do so with oxaloacetic acid since no NADH could be formed from this substrate. It is likely that this oxaloacetic acid decarboxylation activity, as in Lactobacillus plantarum, is distinct from the activity causing the malic-lactic transition. It is also possible that oxaloacetic acid decarboxylation is caused by a malic enzyme. However, there is no verified NAD dependent malic oxidoreductase (decarboxylating) enzyme which does so (12). For example, Macrae (31) isolated a malic enzyme from cauliflower bud mitochondria which showed no activity with oxaloacetic acid. Similarly, Saz (32) isolated a malic enzyme from Ascaris lumbricoides which is also inactive toward oxaloacetic acid. True, the Enzyme Commission (12) lists an enzyme described as L-malate NAD oxidoreductase (decarboxylating) (E.C. 1.1.1.38) which is said to be capable of decarboxylating oxaloacetic acid, but its description dates back to the studies of Ochoa and his group, and we now feel this listing may be improper. 

Antohi (34) has reported several possible isozyme structures of a malic acid decarboxylating enzyme in Bacillus subtilis, and Peak (35) has reported the same in Euglena gracilis this must be kept in mind for the Leuconostoc oenos system. It is possible that the enzymatic activities that we have reported (6,7,8) may be the result of isozyme interactions of the same protein. 

Can we apply any of this information from non-enzymatic catalysis to decarboxylating enzymes ... 

The catalytic power of enzymes is awesome (Table 2.1). A most spectacular example is that of the decarboxylation of orotic acid. It spontaneously decarboxy-lates with tm of 78 million years at room temperature in neutral aqueous solution. Orotidine 5 -phosphate decarboxylase enhances the rate of decarboxylation enzyme-bound substrate by 1017 fold. The classical challenge is to explain the magnitude of the rate enhancements in Table 2.1. We will not ask why enzymatic reactions are so fast but instead examine why the uncatalyzed reactions are so slow, and how they can be speeded up. 

Decarboxylative enzymes may react specifically with amino acids having free polar groups al the a> position. Cadaverine can be produced from lysine, histamine from histidine, and tyramine from tyrosine. 

DE1G Crout, S Davies, RJ Eleath, CO Miles, DR Rathbone, BEP Swoboda. Application of hydrolytic and decarboxylating enzymes in biotransformations. Biocatalysis 9 1-30, 1994. 

Isoquinoline biosynthesis begins with the substrates dopamine and p-hydroxyphenylacetaldehyde (Fig. lb). Dopamine is made from tyrosine by hydroxylation and decarboxylation. Enzymes that catalyze the hydroxylation and decarboxylation steps in either order exist in the plant, and the predominant pathway... 

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). 

Lipoic acid (or thioctic acid) is an essential part of the decarboxylating enzymes... 

In these reactions, the C2-atom of ThDP must be deprotonated to allo v this atom to attack the carbonyl carbon of the different substrates. In all ThDP-dependent enzymes this nucleophilic attack of the deprotonated C2-atom of the coenzyme on the substrates results in the formation of a covalent adduct at the C2-atom of the thiazolium ring of the cofactor (Ila and Ilb in Scheme 16.1). This reaction requires protonation of the carbonyl oxygen of the substrate and sterical orientation of the substituents. In the next step during catalysis either CO2, as in the case of decarboxylating enzymes, or an aldo sugar, as in the case of transketo-lase, is eliminated, accompanied by the formation of an a-carbanion/enamine intermediate (Ilia and Illb in Scheme 16.1). Dependent on the enzyme this intermediate reacts either by elimination of an aldehyde, such as in pyruvate decarboxylase, or with a second substrate, such as in transketolase and acetohydroxyacid synthase. In these reaction steps proton transfer reactions are involved. Furthermore, the a-carbanion/enamine intermediate (Ilia in Scheme 16.1) can be oxidized in enzymes containing a second cofactor, such as in the a-ketoacid dehydrogenases and pyruvate oxidases. In principal, this oxidation reaction corresponds to a hydride transfer reaction. 

The other physiologically important monoamine is 5-hydroxytryptamine (serotonin or 5-HT). It is formed from tryptophan via 5-hydroxytryptophan (5-HTP) Figure 5.2). The nature and properties of tryptophan-5-hydroxy-lase is still obscure, though the hydroxylation of tryptophan in vivo has been demonstrated. There is no clear evidence that this conversion occurs in brain tissue. The decarboxylation of 5-HTP, however, takes place in brain and the decarboxylating enzyme is found in all cerebral areas which contain 5-hydroxytryptamine. 5-HTP decarboxylase is closely related to, if not identical with, DOPA decarboxylase - and agents which inhibit dopan ine formation similarly inhibit the production of 5-hydroxytryptamine. There... 

The classic example of this approach involves the use of levodopa (l-3,4-dihydroxyphenylalanine, Figure 8.13) to treat Parkinson s disease [58]. Parkinson s disease is distinguished by the marked depletion of dopamine— an essential neurotransmitter—in the basal ganglia. Direct dopamine replacement is not possible, because dopamine does not permeate through the blood-brain barrier. However, the metabolic precursor of dopamine, levodopa, is transported across brain capillaries by the neutral amino acid transporter (see Table 5.5 and the related discussion). Peripheral administration of levodopa, therefore, produces an increase in levodopa concentration within the central nervous system some of these molecules are converted into dopamine due to the presence of decarboxylate enzymes in the brain tissue, but decar-boxylate activity is also present in the intestines and blood. To prevent conversion of levodopa into dopamine before entry to the brain, levodopa is usually administered with decarboxylase inhibitors. 

For samples containing several substrates, a preliminary separation should be considered or the total must be determined. In the case of L-amino acid assay the use of decarboxylating enzymes acting selectively on different amino acids is an attractive possibility, and enzyme electrodes of this type are known for L-tyrosine, L-phenylalanine, and L-tryptophan. These sensors will be coupled with a carbon dioxide base sensor. 

Thus, for example, hydrolytically effective enzymes (hydrolases) are distinguished from decarboxylating enzymes (decarboxylases), etc. ... 

Weissbach, H., Lovenberg, W. and Udenfriend, S., Characteristics of mammalian histidine decarboxylating enzymes, Biochim. Biophys. Acta 50, 177 (1961). 

Ganrot, P. O., Rosengren, A. M. and Rosengren, E., On the presence of different histidine decarboxylating enzymes in mammalian tissues, Experientia 17, 263 (1961). 

Brandon (1967) concluded from in vitro experiments that the thermal properties of the carboxylating and decarboxylating enzymes (PEP carboxylase, malic enzyme, see Chap.4.2.3.2) are such that lower temperatures would favor CO2 consumption (i.e., malic acid synthesis) higher temperatures, however, would increase CO2 production by malic acid decarboxylation. Hence, the thermal properties of the enzymic proteins could account partly for the observed temperature effects. 

Many acids undergo decarboxylation. Enzymes that catalyze these reactions are known as carboxylases (EC 6.4.1) and decarboxylases (EC 4.1.1). 

Figure 2.18 The sequence of steps by which an amino-acid attached to pyridoxal and a decarboxylating enzyme is converted to an amine and CO2...

So far we have referred to the decarboxylative enzymes that work on the naturally occurring intermediates of biochemical pathways. Due to the high affinity for the native substrates by these enzymes, however, the applicability to nonnatural synthetic substrates was somewhat limited. Thus we intended to develop a new biocatalysis, such as an enzyme which can decarboxylate a-aryl-a-methylmalonic acids to yield enantiomerically enriched a-substituted a-arylacetic acids because those products are useful compounds as antiinflammatory agents [8-10] and the chiral derivatizing agents [11]. 

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