Carboxyl transfer, from biotin

Some enzymes are nonfunctional until posttranslationally modified. Examples of these enzymes include the acyl- and carboxyltransferases. While lipoate and phosphopantetheine are necessary for acyl transfer chemistry, tethered biotin is used in carboxyl transfer chemistry. Biotin and lipoate tethering occur under a similar mechanism the natural small molecule is activated with ATP to form biotinyl-AMP or lipoyl-AMP (Scheme 20). A lysine from the target protein then attacks the activated acid and transfers the group to the protein. The phosphopantetheine moiety is transferred using its own enzyme, the phosphopantetheinyltrans-ferase (PPTase). The PPTase uses a nucleophilic hydroxy-containing amino acid, serine, to attach the phosphopantetheinyl (Ppant) arm found in coenzyme A to convert the apo (inactive) carrier protein to its holo (active) form. The reaction is Mg -dependent. 

After completion of the first sequence, carboxybiotin moves to the second site, where the carboxyl group is transferred from biotin to pyruvate, forming oxal-oacetate. In essence, a proton and a carboxyl group trade places in this step. Isotope effects indicate that proton removal from pyruvate is not concerted with carboxylation (60, 61). The lack of positional isotope exchange (62) during this process presumably is because the active complex is isolated from solvent, rather than because of a lack of exchangeable sites. 

Acetyl-CoA carboxylase (ACC) catalyzes the first committed step in long-chain fatty acid biosynthesis (see Chapter 7.11). The overall reaction is catalyzed in two sequential reactions (Scheme 3). First, the biotin carboxylase domain catalyzes the ATP-dependent carboxylation of biotin (which is attached to a carrier protein) using bicarbonate as a CO2 donor. In the second reaction, the carboxyl group is transferred from biotin to acetyl-CoA to form malonyl-CoA. In mammals, both reactions are catalyzed by a single protein, but in Escherichia coli and other bacteria, the activity is catalyzed by two separate proteins, a biotin carboxylase and a carboxytransferase. Due to its role in fatty acid synthesis, inhibitors of the overall ACC reaction are proposed to be useful as antiobesity drugs in mammals as well as novel antibiotics against bacteria. 

Nucleophilic attack by biotin on activated bicarbonate forms carboxybiotin. Because the nitrogen of an amide is not nucleophilic, it is likely that the active form of biotin has an enolate-like stmcture. Nucleophilic attack by the substrate (in this case, the enolate form of acetyl-CoA) on carboxybiotin transfers the carboxyl group from biotin to the substrate. 

The initial step involves the formation of carboxybiotinyl enzyme. In the second step, carboxyl transfer from carboxybiotinyl enzyme to an appropriate acceptor substrate takes place, the nature of this acceptor being dependent on the specific enzyme involved. In brief, the function of biotin is to mediate the coupling of ATP cleavage to carboxylation. This is accomplished in two stages in which a carboxybiotin intermediate is formed. In transcarboxylation ATP is not needed because activated carbonate, and not HCO3", is the substrate. 

FIGURE 25.3 In the acetyl-CoA carboxylase reaction, the biotin ring, on its flexible tether, acquires carboxyl groups from carbonylphos-phate on the carboxylase subunit and transfers them to acyl-CoA molecules on the transcarboxylase subunits. 

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]. 

Carboxyphosphate. During the initial carboxyla-tion step lsO from labeled bicarbonate enters the P that is split from ATP. This suggested transient formation of carboxyphosphate by nucleophilic attack of HC03 on ATP (Eq. 14-7). The carboxyl group of this reactive mixed anhydride56 could then be transferred to biotin. This mechanism is supported by the fact that biotin carboxylase catalyzes the transfer of a... 

The activated carboxyl group is then transferred from carboxybiotin to pyruvate to form oxaloacetate. The long, flexible link between biotin and the enzyme enables this prosthetic group to rotate from one active site of the enzyme (the ATP-bicarbonate site) to the other (the pyruvate site). 

All biotin-requiring enzymes follow the same three steps activation of bicarbonate by ATP, reaction of activated bicarbonate with biotin to form carboxybiotin, and transfer of the carboxyl group from carboxybiotin to the substrate. 

Fig. 20.18. Pyruvate carboxylase reaction. Pyruvate carboxylase adds a carboxyl group from bicarbonate (which is in equihbrium with CO2) to pyruvate to form oxaloacetate. Biotin is used to activate and transfer the CO2. The energy to form the covalent biotin-C02 complex is provided by the high-energy phosphate bond of ATP, which is cleaved in the reaction. The enzyme is activated by acetyl CoA.

Biotin (see Scheme 40) is the prosthetic group associated with the biotin-dependent carboxylases and transcarboxylases. The role of biotin is as a carboxyl group carrier. At the first active site, biotin is carboxylated at the N(l) position to form A-carboxybiotin, which acts as CO2 transport. At the second subsite, the carboxyl group is transferred from A-carboxybiotin to substrate. The mechanism of neither half-reaction has been fully defined (for recent discussions see Blonski et al., 1987 Thatcher et ai, 1986). 

Bicarbonate must be activated by the enzyme toward carboxyl transfer. The requirement for ATP and the transfer of labelled oxygen from bicarbonate to inorganic pyrophosphate indicate activation by phosphorylation (Ochoa et ai, 1962). In addition, the most nucleophilic site of biotin is the ureido oxygen. The ureido nitrogen of biotin is a poor nucleophile, and any proposed enzymic mechanism must account for enhancement of the nucleo-philicity of N(T) relative to 0(2 ). 

The mechanistic advantages inherent in the O-phosphobiotin pathway (Scheme 40e) are threefold. First, activation of bicarbonate towards carboxyl transfer is achieved by phosphorylation to form a TBP intermediate from which carboxyl transfer occurs. Secondly, initial phosphorylation of biotin by ATP at the ureido oxygen enhances the nucleophilicity of the N(l ) position. Alternative mechanisms do not provide for activation of the weakly nucleophilic urea nitrogen. Finally, the entropic advantage in the six-membered transition state for carboxyl transfer frees enzyme binding energy that might otherwise have been required to provide correct orientation in a bimolecular reaction. 

Biotin enzymes carboxylases that use biotin as a cofactor. The biotin is bound via an amide bond to the E-amino group of a specific lysine residue in the enzyme protein, i.e. B.e. contain a biotinyllysyl residue. Free (+)-E-V-biotinyl-L-lysine actually occurs in yeast extract, and is known as biocytin. During catalysis, N-atom 1 of the biotin residue is carboxylated in an ATP-dependent reaction ATP + HCOj" + bioti-nyl-enzyme (I) -> ADP + Pj + carboxybiotinyl-en-zyme (II). The carboxyl group is then transferred from (II) to the carboxylase substrate (II) + substrate —> (I) + carboxylated substrate. 

Propionyl CoA is carboxylated by a biotin-containing enzyme (C 3.1) to methylmalonyl CoA. Propionic acid is derived from propionyl CoA by transfer of the CoA residue or by hydrolysis. It is degraded via acrylic acid and lactic acid to pyruvic acid (Fig. 83). 

Biotin (referred to as vitamin H in humans) is an essential cofactor for a number of enzymes that have diverse metabolic functions. Almost a dozen different enzymes use biotin. Among the most well-known are acetyl-CoA carboxylase, pyruvate carboxylase, propionyl-CoA carboxylase, urea carboxylase, methylmalonyl-CoA decarboxylase, and oxaloacetate decarboxylase. Biotin serves as a covalent bound CO2 carrier for reactions in which CO 2 is fixed into an acceptor by carboxylases. Then this carboxyl group in an independent reaction can be transferred from the acceptor substrate to a new acceptor substrate by transcarboxylases, or the carboxyl group can be removed as CO 2 by decarboxylases. 

Basically, biotin behaves as a CO 2-carrier between two sites. Schematically, the biotin carboxylase subsite catalyzes the carboxylation of the biotinyl prosthetic group on the carrier protein. Following the translocation of the carboxylated functional group from the carboxylase subsite to the carboxyl transferase subsite, carboxyl transfer rom 2- lotm to acety-CoA occurs. Presumably free ( + )-biotin can gam access to the carboxylation... 

Two other reactions for succinyl C5oA synthesis may be noted. In heart muscle a CoA transferase is present that requires guanosine triphosphate (GTP) or inosine triphosphate (ITP) but not ATP itaconate can replace succinate in this reaction (54, In animals and bacteria a CoA transferase is found that transfers CoA from acetoacetyl CoA to succinate the enzyme in liver is not highly active. It is thought that in propionic bacteria propionyl CoA is carboxylated with a biotin enzyme to methylmalonyl CoA the reversible conversion of methylmalonyl CoA to succinyl CoA is catalyzed by a Bu enzyme (54a). 

In mammalian tissues, excluding muscle, the most important anaplerotic reaction employs pyruvate carboxylase which contains a biotin prosthetic group (Figure 5.3b) responsible for the transfer of a carboxyl group. ATP provides the energy to bond covalently the carboxyl group from HCO, to the biotin which transfers it when pyruvate binds to the enzyme. In muscle cells, the major pathway utilizes phosphoenolpyruvate and phos-phoenolpyruvate carboxykinase which occurs both in the cytosol and mitochondrial matrix. Two routes are therefore possible in oxaloacetate syn-... 

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