Decarboxylation involving enzymes

Kinetic evidence implicates a pre-association mechanism for catalysis that supports decarboxylation involving reversible formation of a complex of C02 and the carbanionic product.50 The catalyst is able to accelerate the reaction by competing for the carbanion. Such a situation would routinely be available in an enzyme active site.37 The complex cannot be observed spectroscopically because of its short lifetime and low concentration. However, catalysis after C C bond-breaking should alter the observed 12C/13C kinetic isotope effects (CKIE). 

Recent model studies strongly support the proposed mechanism. The first crystal structures of Fe(II) complexed to benzoylformate show that an a-keto acid can coordinate to the iron as either a monodentate or didentate ligand [236]. Exposure of these [Fe(II)(L)(bf)]+ complexes (L = tmpa or 6-Me3-tmpa) to 02 results in the quantitative conversion of benzoylformate to benzoic acid and C02, modeling the oxidative decarboxylation reaction characteristic of this class of enzymes. As with the enzymes, the use of 1802 in the model studies results in the incorporation of the label into the benzoate product. For [Fe(6-Me3-tmpa)(bf)]+, the rate of the oxidative decarboxylation increases as the substituent of the benzoylformate becomes more electron-withdrawing, affording a Hammett p of +1.07. This suggests that the oxidative decarboxylation involves a nucleophilic attack, most plausibly by the iron-bound 02, on the keto carbon of benzoylformate to initiate decarboxylation as proposed in Figure 27. 

Many unusual biocatalytic asymmetric oxidation reactions like oxidative cychza-tion, oxidative ring expansion, oxidative deamination, or oxidative decarboxylation were discovered in the course of studies in natural product biosynthesis and the involved enzyme functions continue to be of great interest. 

Isocitrate dehydrogenase catalyzes the first of two decarboxylations and dehydrogenations in the cycle. Three different isocitrate dehydrogenases are present one specific for NAD+ and found only in mitochondria, the other two specific for NADP+ and found in mitochondria and cytoplasm. The NAD -specific enzyme is the primary enzyme with regard to TCA cycle operation. All three require Mg + or Mn +. The reaction yields a-ketoglutarate (2-oxoglutarate), NAD(P)H, and CO2 and involves enzyme-bound oxalosuccinate as an intermediate. 

Most carboxylations and decarboxylations involve anionic intermediates [cf. Eq. (2)]. The stability of the anion is an important factor in determining reaction rates. An unstable anion will be very reactive toward CO2, but it will be difficult to form. A stable anion will be less reactive toward CO2, but it will be easy to form. Many carboxylations involve removal of a proton from an acid of pKa 15-18. Enzymic stabilization of such anions is very important. 

Carboxylation of RuBP to form the six-carbon intermediate is significantly endergonic, whereas the overall reaction is exergonic by about 6 kcal/mol. Overall, the reaction is driven by cleavage of the six-carbon intermediate. Clearly the enzyme controls the conformation of the six-carbon intermediate in a way that favors cleavage and disfavors decarboxylation. The enzyme from R. rubrum is less successful at this than the more complex higher plant enzyme. Metal com-plexation is probably a key to control, and the small subunit might also be involved. 

Biotin is involved in carboxylation and decarboxylation reactions. It is covalently bound to its enzyme. In the carboxylase reaction, C02 is first attached to biotin at the ureido nitrogen, opposite the side chain in an ATP-dependent reaction. The activated C02 is then transferred from carboxybiotin to the substrate. The four enzymes of the intermediary metabolism requiring biotin as a prosthetic group are pyruvate carboxylase (pyruvate oxaloacetate), propionyl-CoA-carboxylase (propionyl-CoA methylmalonyl-CoA), 3-methylcroto-nyl-CoA-carboxylase (metabolism of leucine), and actyl-CoA-carboxylase (acetyl-CoA malonyl-CoA) [1]. 

The synthesis and metabolism of trace amines and monoamine neurotransmitters largely overlap [1]. The trace amines PEA, TYR and TRP are synthesized in neurons by decarboxylation of precursor amino acids through the enzyme aromatic amino acid decarboxylase (AADC). OCT is derived from TYR. by involvement of the enzyme dopamine (3-hydroxylase (Fig. 1 DBH). The catabolism of trace amines occurs in both glia and neurons and is predominantly mediated by monoamine oxidases (MAO-A and -B). While TYR., TRP and OCT show approximately equal affinities toward MAO-A and MAO-B, PEA serves as preferred substrate for MAO-B. The metabolites phenylacetic acid (PEA), hydroxyphenylacetic acid (TYR.), hydroxymandelic acid (OCT), and indole-3-acetic (TRP) are believed to be pharmacologically inactive. 

TPP-dependent enzymes are involved in oxidative decarboxylation of a-keto acids, making them available for energy metabolism. Transketolase is involved in the formation of NADPH and pentose in the pentose phosphate pathway. This reaction is important for several other synthetic pathways. It is furthermore assumed that the above-mentioned enzymes are involved in the function of neurotransmitters and nerve conduction, though the exact mechanisms remain unclear. 

The conversion of tyrosine to epinephrine requires four sequential steps (1) ring hydroxylation (2) decarboxylation (3) side chain hydroxylation to form norepinephrine and (4) N-methylation to form epinephrine. The biosynthetic pathway and the enzymes involved are illustrated in Figure 42-10. 

Pyridoxal phosphate is a coenzyme for many enzymes involved in amino acid metabolism, especially in transamination and decarboxylation. It is also the cofactor of glycogen phosphorylase, where the phosphate group is catalytically important. In addition, vitamin Bg is important in steroid hormone action where it removes the hormone-receptor complex from DNA binding, terminating the action of the hormones. In vitamin Bg deficiency, this results in increased sensitivity to the actions of low concentrations of estrogens, androgens, cortisol, and vitamin D. 

A number of iron-containing, ascorbate-requiring hydroxylases share a common reaction mechanism in which hydroxylation of the substrate is linked to decarboxylation of a-ketoglutarate (Figure 28-11). Many of these enzymes are involved in the modification of precursor proteins. Proline and lysine hydroxylases are required for the postsynthetic modification of procollagen to collagen, and prohne hydroxylase is also required in formation of osteocalcin and the Clq component of complement. Aspartate P-hydroxylase is required for the postsynthetic modification of the precursor of protein C, the vitamin K-dependent protease which hydrolyzes activated factor V in the blood clotting cascade. TrimethyUysine and y-butyrobetaine hydroxylases are required for the synthesis of carnitine. 

Biogenic amines are decarboxylated derivatives of tyrosine and tryptophan that are found in animals from simple invertebrates to mammals. These compounds are found in neural tissue, where they function as neurotransmitters, and in non-neural tissues, where they have a variety of functions. The enzymes involved in biogenic amine synthesis and many receptors for these compounds have been isolated from both invertebrate and vertebrate sources. In all cases, the individual proteins that effect biogenic amine metabolism and function show striking similarity between species, indicating that these are ancient and well-conserved pathways. 

Hydrocarbon formation involves the removal of one carbon from an acyl-CoA to produce a one carbon shorter hydrocarbon. The mechanism behind this transformation is controversial. It has been suggested that it is either a decarbonylation or a decarboxylation reaction. The decarbonylation reaction involves reduction to an aldehyde intermediate and then decarbonylation to the hydrocarbon and releasing carbon monoxide without the requirement of oxygen or other cofactors [88,89]. In contrast, other work has shown that acyl-CoA is reduced to an aldehyde intermediate and then decarboxylated to the hydrocarbon, releasing carbon dioxide [90]. This reaction requires oxygen and NADPH and is apparently catalyzed by a cytochrome P450 [91]. Whether or not a decarbonylation reaction or a decarboxylation reaction produces hydrocarbons in insects awaits further research on the specific enzymes involved. 

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