Reactions of the Citric Acid Cycle:- Biotin in Pyruvate Carboxylase Carries CO2 Groups
The pyruvate carboxylase reaction requires the vitamin biotin (Fig. 16–16), which is the prosthetic group of the enzyme. Biotin plays a key role in many carboxylation reactions. It is a specialized carrier of one-carbon groups in their most oxidized form: CO2. (The transfer of one-carbon groups in more reduced forms is mediated by other cofactors, notably tetrahydrofolate and S-adenosylmethionine, Carboxyl groups are activated in a reaction that splits ATP and joins CO2 to enzyme-bound biotin. This “activated” CO2 is then passed to an acceptor (pyruvate in this case) in a carboxylation reaction. Pyruvate carboxylase has four identical subunits, each containing a molecule of biotin covalently attached through an amide linkage to the ε-amino group of a specific Lys residue in the enzyme active site. Carboxylation of pyruvate proceeds in two steps (Fig. 16–16): first, a carboxyl group derived from HCO3 is attached to biotin,
MECHANISM FIGURE 16–16 The role of biotin in the reaction cat alyzed by pyruvate carboxylase. Biotin is attached to the enzyme through an amide bond with the -amino group of a Lys residue, forming biotinyl-enzyme. Biotin-mediated carboxylation reactions occur in two phases, generally catalyzed by separate active sites on the en zyme as exemplified by the pyruvate carboxylase reaction. In the first phase (steps 1 to 3), bicarbonate is converted to the more activated CO2, and then used to carboxylate biotin. The bicarbonate is first activated by reaction with ATP to form carboxyphosphate (step 1), which breaks down to carbon dioxide (step 2). In effect, the bicarbonate is dehydrated by its reaction with ATP, and the CO2 can react with biotin to form carboxybiotin (step 3). The biotin acts as a carrier to transport the CO2 from one active site to another on the same enzyme (step 4). In the second phase of the reaction (steps 5 to 7), catalyzed in a second active site, the CO2 reacts with pyruvate to form oxaloacetate. The CO2 is released in the second ac tive site (step 5). Pyruvate is converted to its enolate form in step 6, transferring a proton to biotin. The enolate then attacks the CO2 to generate oxaloacetate in the final step of the reaction (step 7).
then the carboxyl group is transferred to pyruvate to form oxaloacetate. These two steps occur at separate active sites; the long flexible arm of biotin transfers activated carboxyl groups from the first active site to the second, functioning much like the long lipoyllysine arm of E2 in the PDH complex (Fig. 16–6) and the long arm of the CoA-like moiety in the acyl carrier protein involved in fatty acid synthesis (see Fig. 21–5); these are compared in Figure 16–17. Lipoate, biotin, and pantothenate all enter cells on the same transporter; all become covalently attached to proteins by similar reactions; and all provide a flexible tether that allows bound reaction intermediates to move from one active site to another in an enzyme complex, without dissociating from it—all, that is, participate in substrate channeling. Biotin is a vitamin required in the human diet; it is abundant in many foods and is synthesized by intestinal bacteria. Biotin deficiency is rare, but can sometimes be caused by a diet rich in raw eggs. Egg whites contain a large amount of the protein avidin (Mr 70,000), which binds very tightly to biotin and prevents its absorption in the intestine. The avidin of egg whites may be a defense mechanism for the potential chick embryo, inhibiting the growth of bacteria. When eggs are cooked, avidin is denatured (and thereby inactivated) along with all other egg white proteins. Purified avidin is a useful reagent in biochemistry and cell biology. A protein that contains covalently bound biotin (derived experimentally or produced in vivo) can be recovered by affinity chromatography (see Fig. 3–18c) based on biotin’s strong affinity for avidin. The protein is then eluted from the column with an excess of free biotin. The very high affinity of biotin for avidin is also used in the laboratory in the form of a molecular glue that can hold two structures together.
FIGURE 16–17 Biological tethers. The cofactors lipoate, biotin, and the combination of -mercaptoethylamine and pantothenate form long, flexible arms in the enzymes to which they are covalently bound, acting as tethers that move intermediates from one active site to the next. The group shaded pink is in each case the point of attachment of the activated intermediate to the tether.
