Biotin-dependent carboxylations

We have briefly noted the role of biotin when we considered the biosynthesis of fatty acids (see Section 15.5). Biotin is a carrier of carbon dioxide and involved in carboxylation reactions. In fatty acid biosynthesis, we noted how acetyl-CoA was 

Carbon dioxide is a normally unreactive material, and combination with biotin requires the input of energy (from ATP). Carbon dioxide is usually present 

This mixed anhydride carboxylates the coenzyme in a biotin-enzyme complex. Biotin is bound to a lysine residue in the enzyme as an amide. The carboxylation 

In what can be considered a reversal of this sequence, the acetyl-CoA acts as the nueleophile and is carboxylated to malonyl-CoA with displacement of the biotin-enzyme system. 

Fixation of carbon dioxide by biotin-enzyme complexes is not unique to acetyl-CoA, and another important example occurs in the generation of oxaloacetate from pyravate in the synthesis of glucose from non-carbohydrate sources (gluconeogene-sis). This reaction also allows replenishment of Krebs 

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Most biotin-dependent carboxylations (Table 18.5) use bicarbonate as the carboxylating agent and transfer the carboxyl group to a substrate earbanion. Bicarbonate is plentiful in biological fluids, but it is a poor electrophile at carbon and must be activated for attack by the substrate earbanion. 

O Pyruvate undergoes a biotin-dependent carboxylation on the methyl group to give oxaloacetate... 

Knowles, J. R., The mechanism of biotin-dependent enzymes. Ann. Rev. Biochem. 58 195, 1989. Review of the chemical mechanism of biotin-dependent carboxylation reactions. 

The main metabolic function of vitamin K is as the coenzyme in the carboxyla-tion ofprotein-incorporated glutamate residues to yield y -carboxyglutamate -a unique type of carboxylation reaction, clearly distinct from the biotin-dependent carboxylation reactions (Section 11.2.1). 

Figure 29.6 MECHANISM Mechanism of step 3 in Figure 29.5, the biotin-dependent carboxylation of acetyl CoA to yield malonyl CoA.

FIGURE 9,32 Structure of biotin. Biotin is used as the carrier of carboxyl groups in carboxyialion reactions. In an ATl -dcpendcnt reaction, bicarbonate is transferred to biotin, generating carboxy-biotin. The carboxyl group is bound to the I -nitrogpn. The second half of biotin-dependent carboxylation reactions involves transfer of the carboxyl group to the substrate. 

STEP 1 P3mivate undergoes biotin-dependent carboxylation to give oxaloacetate. 

Biotin (Fig. 8.40) is essential, a normal constituent of the diet, and required for four biotin-dependent carboxylation reactions. Eating raw egg white can induce a deficiency. 

The committed (rate-controlling) step is the biotin-dependent carboxylation of acetyl-CoA by acetyl-CoA carboxylase. Important allosteric effectors are citrate (positive) and long-chain acyl-CoA derivatives (negative). 

The formation of a bond between the carboxylate group derived from bicarbonate and a carbon adjacent to a carbonyl group is indicative of a reaction catalyzed by an enzyme that utilizes biotin as a cofactor (Scheme 16). The recent review by Knowles covers many recent discoveries relating to the role of enzymes in these reactions (43). (Editor s note For additional aspects of biotin-dependent carboxylation, see Chapter 6 by O Leary.) Most biotin-dependent enzymes promote a two-step process in which Ai-carboxybiotin serves as an intermediate in a process involving the exchange of the caiix)xylate group derived from bicarbonate for a proton at the a-carbon of the carbonyl compound (44). 

After dehydrogenation to 234, X = SCoA, the catabolism of leucine 205 (Scheme 62c) differs from that of the other branched-chain amino acids. A biotin-dependent carboxylation leads to the acid 236, X = SCoA, which is hydrated to HMG-CoA 237, a compound involved in isoprenoid biosynthesis. Feeding stereospecifically labeled samples of leucine in studies of terpenoid biosynthesis indicated that the ( )-methyl group was carboxylated without isomerization of the double bond (181, 182). Messner, Cornforth et al. (215) investigated the hydration 236 = 237 catalyzed by the enzyme 3-methyl-glutaconyl-CoA hydratase (EC 4. 2. 1. 18) and showed that the reversible reaction had syn stereospecificity. 

This COa transfer is of interest because some biological oxidations are known to involve such reactions (e.g ., biotin-dependent carboxylations). 

Malonyl-CoA is synthesized in a biotin-dependent carboxylation of acetyl-CoA (see Biotin, under Vitamins) ... 

Proof that a carboxylated en2yme intermediate (enzyme-COf) actually participates in biotin-dependent carboxylations was provided by Yoshito Kaziro in Severn Ochoa s laboratory at New York University. They were able to isolate enzyme-C02 and show an exact stoichiometry between bound biotin and the active carboxy group. Importantly, the isolated enzyme-COr transferred its labile carboxy group to propionyl-CoA yielding methylmalonyl-CoA [reaction (3)] or underwent quantitative decarboxylation upon exposure to ADP, P and Mg + [reverse of reaction (2)]. 

FIGURE 23.7 M ECHAN ISM Mechanism of step 3 in Figure 23.6, the biotin-dependent carboxylation of acetyl CoA to yield malonyl CoA. This mechanism is essentially identical to that shown previously in Figure 22.10 for the carboxylation of pyruvate in gluconeogenesis. 

However, since it is not known to what extent the enzyme contributes to the stabilization of the leaving group anion X in the biotin-dependent carboxylation reactions, a conclusion cannot be drawn a priori as to whether O- or N-attack occurs. Clearly, simple model compounds are not always reliable indicators of reactivity in the environment of an enzyme (343). Maybe enzyme-bound biotin reacts in its high energy isourea form to increase the nucleophilicity of the nitrogen atom. After all an imido nitrogen is known to be a better nucleophile than an amido nitrogen. 

Like other biotin-dependent carboxylations, this reaction involves the abstraction of the a-proton (tritium here) and its replacement by CO2. This aspect was also analyzed by R6tey and Lynen (336) and by Rose and co-workers (345). They proposed that proton abstraction and carboxylation occur in a concerted process (Fig. 7.18). 

Lynen, F., 1967, The role of biotin-dependent carboxylations in biosynthetic reactions, Biochem. J. 102 381. 

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