Enzymes, decarboxylation

D-Mevalonic acid is the fundamental intermediate in the biosynthesis of the terpenoids and steroids, together classed as poly-isoprenoids. The biogenetic isoprene unit is isopentenyl pyrophosphate which arises by enzymic decarboxylation-dehydration of mevalonic acid pyrophosphate. D-Mevalonic acid is almost quantitatively incorporated into cholesterol synthesized by rat liver homogenates. 

The result of enzymatic decarboxylation was extremely clear. While (S)-compound resulted in C-containing product, (/ )-compound gave the product with C no more than natural abundance. Apparently, the enzyme decarboxylated pro-(/ ) carboxyl group selectively and the reaction proceeds with net inversion of configuration. Thus, the presence of a planar intermediate can be reasonably postulated. Enantioface-differentiating protonation to the intermediate will give the optically active final product (Eig. 12). 

Enzymic Decarboxylation of an a,/S-Acetylenic Acid. J. Chem. Soc. [London] 1961, 1532. 

The only known substrate for enzymic decarboxylation is uridine 5 -(a-D-glucopyranosyluronic acid pyrophosphate). A simple reaction of this type, ieading to the a-D-xylopyranosyl ester, has been ob-... 

Hamilton marked the carbonyl group of acetoacetic acid with ieO, and then carried out the enzymic decarboxylation (Hamilton and Westheimer, 1959). The product of the decarboxylation, acetone, contained none of the label. This result is demanded by the ketimine mechanism, whereas the mechanism of uncatalyzed decarboxylation would have required that the label appear intact in the product. Of course, in order to make these statements we had to carry out an elaborate set of control experiments, since 180 is washed out of both acetone and acetoacetic acid by buffers and even more... 

A model system and mechanism (12) had also been developed for the metal-ion promoted enzymic decarboxylations of dibasic ketoacids, such as oxaloacetic acid (Steinberger and Westheimer, 1949,1951). 

In enzymic decarboxylations the mechanistic pathway seems to involve Schiff base formation between an —NH2 from a lysine residue and a C=0 of the keto acid.52 Likewise, with small-molecule primary amines, catalysis of decarboxylation of /3-ketoacids53-58 has been ascribed to a Schiff base intermediate. The overall reaction for oxalacetate is... 

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). 

Both bacteria and plants have separate enzymes that catalyze the individual steps in the biosynthetic sequence (Fig. 17-12). The fatty acyl group grows while attached to the small acyl carrier protein (ACP).54 58 Control of the process is provided, in part, by the existence of isoenzyme forms. For example, in E. coli there are three different P-oxoacyl-ACP synthases. They carry out the transfer of any acyl primer from ACP to the enzyme, decarboxylate malonyl-ACP, and carry out the Claisen condensation (steps b, e, and/in Eq. 17-12)58a e One of the isoenzymes is specialized for the initial elongation of acetyl-ACP and also provides feedback regulation.58c The other two function specifically in synthesis of unsaturated fatty acids. 

An especially interesting coenzyme is thiamine pyrophosphate (vitamin Bx) which, in conjunction with the appropriate enzyme, decarboxylates 2,-oxopropanoic acid (pyruvic acid Section 20-1 OB). We can write the overall reaction as follows ... 

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... 

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). 

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... 

Heavy-atom isotope effects for the spontaneous and enzymic decarboxylations are large and indicate that in both cases, the decarboxylation step is fully rate determining. The near equality of isotope effects in the two cases suggests that transition states for the two reactions are similar (24, 25). 

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. 

Compulsory oxygen-18 exchange from the carbonyl group of the substrate accompanies the enzymic decarboxylation (95). Reaction of the enzyme with acetoacetate in the presence of NaBHa results in inactivation of the enzyme and formation of a product in which a single lysine amino group has been alkylated (96). This and a variety of other lines of evidence indicate that the reaction occurs by means of a Schiff base mechanism analogous to the amine-catalyzed decarboxylation (Scheme VII). Except for the reactive lysine, the identities of other catalytic groups at the active site are not known. 

The rate of the enzymic decarboxylation of acetoacetic acid exceeds the rate of the spontaneous reaction by perhaps a billionfold. However, simple amines of appropriate pK also catalyze the reaction, and the difference in rate between catalysis by the enzyme and catalysis by cyanomethylamine (pK 5.5) is only about 100-fold (]00). 

The decarboxylation is more enigmatic. Although models for Schiff base formation and transamination are common, models for decarboxylation are rare. If PLP is mixed with an amino acid in aqueous solution, then Schiff base formation will occur, followed by slow transamination. No decarboxylation is observed. If an a-methyl amino acid is used, thus preventing transamination, slow decarboxylation is observed, but only at temperatures above 100°C (106). Thus, the problem in enzymic decarboxylation is to catalyze a very slow reaction and avoid a much more favorable reaction. 

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 above general mechanism for non-enzymic, pyridoxal-catalysed processes was derived mainly from a study of transamination reactions. Nevertheless, non-enzymic, pyridoxal-catalysed decarboxylations have been reported, for example, that of histidine to histamine. The following pyridoxal-catalysed, non-enzymic decarboxylations of a-aminoisobutyric acid (R = = GHg), a-methylserine (R = GHjOH R = GHg) and... 

The first reaction is equivalent to enzymic decarboxylation however, no enzymic equivalent of the second reaction is known. Both were inhibited by the presence of those metal ions which catalyse the other possible pathways covered by the general scheme Figure 4.2). When salicylaldehyde XVIII) or 4-formyl-5-hydroxymethyl-3-methoxy-2-methylpyridine XIX) was used... 

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. 

Figure 4.3. Mechanism for the enzymic decarboxylation of amino acids proposed by Werle and Koch. For histidine,...

Like most of the PLP-dependent decarboxylases, these enzymes involve retention of configuration at the a-carbon (Fig. 13). Chang and Snell first observed that histidine decarboxylase catalyzes the conversion of L-histidine to histamine in solvent with the stereospecific incorporation of one deuterium atom from H20 solvent (266). Retention of configuration was tentatively suggested on the basis of a comparison of the optical rotation properties of the deuterated histamine with those of model compounds. This conclusion was later confirmed for histidine decarboxylase from both Lactobacillus 30a and Clostridium welchii by a method that employed diamine oxidase for the configurational analysis of the (a5)-[a- H]histamine resulting from enzymic decarboxylation of (aS)-[of- H]histidine in H2O diamine oxidase catalyzes stereospecific removal of the pro- S) hydrogen at the a-methylene of histamine (267, 2 ) [Eq. (53)] ... 

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