Lipoate

Pyruvate and a-ketoglutarate dehydrogenase have complex systems involving lipoate and FAD prior to the passage of electrons to NAD, while electron trans-... 

Recently, Prasad et al. cloned a mammalian Na+-dependent multivitamin transporter (SMVT) from rat placenta [305], This transporter is very highly expressed in intestine and transports pantothenate, biotin, and lipoate [305, 306]. Additionally, it has been suggested that there are other specific transport systems for more water-soluble vitamins. Takanaga et al. [307] demonstrated that nicotinic acid is absorbed by two independent active transport mechanisms from small intestine one is a proton cotransporter and the other an anion antiporter. These nicotinic acid related transporters are capable of taking up monocarboxylic acid-like drugs such as valproic acid, salicylic acid, and penicillins [5], Also, more water-soluble transporters were discovered as Huang and Swann [308] reported the possible occurrence of high-affinity riboflavin transporter(s) on the microvillous membrane. 

Prasad, P. D., et al. Cloning and functional expression of a cDNA encoding a mammalian sodium-dependent vitamin transporter mediating the uptake of pantothenate, biotin, and lipoate. J. Biol. Chem. 1998, 273, 7501-7506. 

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. 

Scheme 22 Mechanism for tethered lipoate. Lipoate is tethered to enzymes and used as acyi-hoider for muiticomponent synthetic pathways.

Silver M, Kelly DP. 1976b. Thin layer chromatography of oxidised and reduced lipoate and lipoamide and their persulfides. J Chromatog 123 479-81. 

Figure 3.4 Structure of two prosthetic groups (a) biotin (b) lipoate. Biotin functions as a carboxyl group carrier, e.g. in acetyl-CoA carboxylase. Lipoate is presented in its oxidised form (-S-S-). It is a cofactor for pyruvate dehydrogenase and oxoglu-tarate dehydrogenase.

Lipoate Electrons and acyl groups Not required in diet... 

The combined dehydrogenation and decarboxylation of pyruvate to the acetyl group of acetyl-CoA (Fig. 16-2) requires the sequential action of three different enzymes and five different coenzymes or prosthetic groups—thiamine pyrophosphate (TPP), flavin adenine dinucleotide (FAD), coenzyme A (CoA, sometimes denoted CoA-SH, to emphasize the role of the —SH group), nicotinamide adenine dinucleotide (NAD), and lipoate. Four different vitamins required in human nutrition are vital components of this system thiamine (in TPP), riboflavin (in FAD), niacin (in NAD), and pantothenate (in CoA). We have already described the roles of FAD and NAD as electron carriers (Chapter 13), and we have encountered TPP as the coenzyme of pyruvate decarboxylase (see Fig. 14-13). 

The fifth cofactor of the PDH complex, lipoate (Fig. 16-4), has two thiol groups that can undergo reversible oxidation to a disulfide bond (—S—S—), similar to that between two Cys residues in a protein. Because of its capacity to undergo oxidation-reduction reactions, lipoate can serve both as an electron hydrogen carrier and as an acyl carrier, as we shall see. 

FIGURE 16-4 Lipoic acid (lipoate) in amide linkage with a Lys residue. The lipoyllysyl moiety is the prosthetic group of dihydrolipoyl transacetylase (E2 of the PDH complex). The lipoyl group occurs in oxidized (disulfide) and reduced (dithiol) forms and acts as a carrier of both hydrogen and an acetyl (or other acyl) group. 

Ei catalyzes first the decarboxylation of pyruvate, producing hydroxyethyl-TPP, and then the oxidation of the hydroxyethyl group to an acetyl group. The electrons from this oxidation reduce the disulfide of lipoate bound to E2, and the acetyl group is transferred into thioester linkage with one —SH group of reduced lipoate. 

E3 catalyzes the regeneration of the disulfide (oxidized) form of lipoate electrons pass first to FAD, then to NAD+. 

FIGURE 16-17 Biological tethers. The cofactors lipoate, biotin, and the combination of /3-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. 

Review of the roles of swinging arms containing lipoate, biotin, and pantothenate in substrate channeling through multienzyme complexes. 

Mixed acid fermentations are not limited to bacteria. For example, trichomonads, parasitic flagellated protozoa, have no mitochondria. They export pyruvate into the bloodstreams of their hosts and also contain particles called hydrogenosomes which can convert pyruvate to acetate, succinate, C02, and H2.144 Hydrogenosomes are bounded by double membranes and have a common evolutionary relationship with both mitochondria and bacteria. The enzyme that catalyzes pyruvate cleavage in hydrogenosomes apparently does not contain lipoate and may be related to the pyruvate-ferredoxin oxidoreductase of clostridia (Eq. 15-35). The hydrogenosomes also contain an active hydrogenase. 

There are two 2-oxoacid dehydrogenase multienzyme complexes in E. coli. One is specific for pyruvate, the other for 2-oxoglutarate. Each complex is about the size of a ribosome, about 300 A across. The pyruvate dehydrogenase is composed of three types of polypeptide chains El, the pyruvate decarboxylase (an a2 dimer of Mr — 2 X 100 000) E2, lipoate acetyltransferase (Mr = 80 000) and E3, lipoamide dehydrogenase (an a2 dimer of Mr = 2 X 56 000). These catalyze the oxidative decarboxylation of pyruvate via reactions 1.6, 1.7, and 1.8. (The relevant chemistry of the reactions of thiamine pyrophosphate [TPP], hydroxyethylthiamine pyrophosphate [HETPPJ, and lipoic acid [lip-S2] is discussed in detail in Chapter 2, section C3.)... 

With lipoate as acceptor t.2.99 With other acceptors... 

An intramolecular model for the reductive acyl transfer catalysed by a-keto-acid dehydrogenases relies on the presence of PhHgCl to trap the thiolate generated by reduction of the lipoate disulfide bond by enamine.280 This shows a 10-fold increase in loss of enamine UV-visible absorption over background decomposition attributed by the authors to reductive acyl transfer. However, no reaction products were isolated and... 

Fuchs, J. and Milbradt, R., Antioxidant inhibition of skin inflammation induced by reactive oxidants evaluation of the redox couple dihydrolipoate/lipoate, Skin Pharmacol., 1, 278, 1994. 

Answer Oxidative decarboxylation involving NADP+ or NAD+ as the electron acceptor the a-ketoglutarate dehydrogenase reaction is also an oxidative decarboxylation, but its mechanism is different and involves different cofactors TPP, lipoate, FAD, NAD+, and CoA-SH. 

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