Lipoamide reduced form

Lipoic acid exists as a mixture of two structures a closed-ring disulfide form and an open-chain reduced form (Figure 18.33). Oxidation-reduction cycles interconvert these two species. As is the case for biotin, lipoic acid does not often occur free in nature, but rather is covalently attached in amide linkage with lysine residues on enzymes. The enzyme that catalyzes the formation of the lipoamide nk.2Lg c requires ATP and produces lipoamide-enzyme conjugates, AMP, and pyrophosphate as products of the reaction. 

Figure 2. Mechanism of PDH. The three different subunits of the PDH complex in the mitochondrial matrix (E, pyruvate decarboxylase E2, dihydrolipoamide acyltrans-ferase Ej, dihydrolipoamide dehydrogenase) catalyze the oxidative decarboxylation of pyruvate to acetyl-CoA and CO2. E, decarboxylates pyruvate and transfers the acetyl-group to lipoamide. Lipoamide is linked to the group of a lysine residue to E2 to form a flexible chain which rotates between the active sites of E, E2, and E3. E2 then transfers the acetyl-group from lipoamide to CoASH leaving the lipoamide in the reduced form. This in turn is oxidized by E3, which is an NAD-dependent (low potential) flavoprotein, completing the catalytic cycle. PDH activity is controlled in two ways by product inhibition by NADH and acetyl-CoA formed from pyruvate (or by P-oxidation), and by inactivation by phosphorylation of Ej by a specific ATP-de-pendent protein kinase associated with the complex, or activation by dephosphorylation by a specific phosphoprotein phosphatase. The phosphatase is activated by increases in the concentration of Ca in the matrix. The combination of insulin with its cell surface receptor activates PDH by activating the phosphatase by an unknown mechanism.

The stability of EH2 is very species dependent. All of the above results refer to the pig heart enzyme and, where tested, to other mammalian species. It was initially reported that no long wavelength absorption was observed upon reduction of E. coli enzyme with NADH 109), but reduction by 1 equivalent of NADH or dihydrolipoamide leads to the formation of 25% of the maximal 2-electron-reduced species 108) and similar results are obtained with the Azotobacter enzyme 114)- That this species is the catalytically important one in the E. coli enzyme as well as in the mammalian enzyme has also been demonstrated 50). Reduction with dihydrolipoamide in the rapid reaction spectrophotometer at 2° results in the full formation of EH2 followed by the slow k = 13 min, 1 mAf dihydrolipoamide) four-electron reduction. The spectrum of EHa generated in this way is shown in Fig. 7 and is identical with that of the pig heart enzyme. The 2-electron-reduced form, EHj of lipoamide dehydrogenase of spinach 99) may be somewhat unstable however, spectrally it is difficult to distinguish between instability and formation of the EHa-NADH complex (see above) on the basis of available spectral data. Either phenomenon could lead to inhibition by excess NADH. In glutathione reductase it is possible that the complex can be rapidly reoxidized by glutathione 53). 

Thiamin binds the oxo-acid substrate, decarboxylating it to an active aldehyde intermediate. This is then transferred to enzyme-bound lipoamide, reducing the disulfide bridge of the lipoamide and forming a thioester. The resultant acyl group is transferred to coenzyme A (CoA), and the dithiol lipoamide is reoxidized hy NAD+. 

Other reagents recommended for the reduction of isoxazoles to 1,3-enaminoketones include samarium diiodide, molybdenum hexacarbonyl in aqueous acetonitrile, and diiron nonacarbonyl in water. Electrochemical methods have also been employed." A particularly mild method involves reaction with the reduced form of the coenzyme lipoamide (LA2) complexed with iron(II). Birch reduction (sodium and... 

Fio. 6. Complex formation between the 2-electron-reduced form of pig heart lipoamide dehydrogenase and NAD 118). 

Dihydrolipoic acid or dihydrolipoamide The two-electron reduced form of lipoic acid or lipoamide containing... 

Figure 11.2 Reaction sequences catalyzed by 2-oxoacid dehydrogenase complex Pyruvate dehydrogenase complex (PDC) and a-ketoglutarate dehydrogenase complex (aKGDC) catalyze the oxidative decarboxylation of pyruvate (R = CH3) and a-ketoglutarate (R = CH2CH2COOH) to Acetyl-CoA and succinyl CoA respectively. Three component enzymes 2-oxoacid (pyruvate/a-ketoglutarate) decarboxylase, lipoate acetyltransferase/succinyltransferase, dihydrolipoate dehydrogenase as well as five cofactors, namely (1) thiamine pyrophosphate (TPP) and its acylated form, (2) lipoamide (LipS2), reduced form and acylated form, (3) flavin adenine dinucleotide (FAD) and its reduced form, (4) nicotinamide adenine dinucleotide (NAD ) and its reduced form, and (5) coenzyme A (CoASH) and its acylated product are involved.

Figure 7-2. Reactions of the pyruvate dehydrogenase (PDU) multienzyme complex (PDC). Pyruvate is decarboxylated by the PDH subunit (I ,) in the presence of thiamine pyrophosphate (TPP). The resulting hydroxyethyl-TPP complex reacts with oxidized lipoamide (LipS3), the prosthetic group of dehydrolipoamide transacetylase (Ii2), to form acetyl lipoamide. In turn, this intermediate reacts with coenzyme A (CoASH) to yield acetyl-CoA and reduced lipoamide [Lip(SH)2]. The cycle of reaction is completed when reduced lipoamide is reoxidized by the flavoprotein, dehydrolipoamide dehydrogenase (E3). Finally, the reduced flavoprotein is oxidized by NAD+ and transfers reducing equivalents to the respiratory chain via reduced NADH. PDC is regulated in part by reversible phosphorylation, in which the phosphorylated enzyme is inactive. Increases in the in-tramitochondrial ratios of NADH/NAD+ and acetyl-CoA/CoASH also stimulate kinase-mediated phosphorylation of PDC. The drug dichloroacetate (DCA) inhibits the kinase responsible for phosphorylating PDC, thus locking the enzyme in its unphosphory-lated, catalytically active state. Reprinted with permission from Stacpoole et al. (2003).

Lipoamide dehydrogenase is quite stable in 6.5 M urea provided it is not frozen or reduced full activity is regained upon dilution (32, 170). However, if reduced with NADH in 6.5 Af urea, EH, which is initially formed, is further reduced over a period of a few hours. The activity remaining upon dilution during this period is directly proportional to the amount of EHz remaining at the time of dilution. NAD stabilizes EHz in 6.5 M urea just as it does in the absence of urea. When reduction in urea is allowed to go to completion, the spectrum upon reoxidation is that of free FAD indicating dissociation as a result of reductive denaturation [82, 170). 

The lack of reactivity of the semiquinone per se with either thioredoxin or NADPH shows that it cannot be involved in catalysis. The rapid production of semiquinone by irradiation of partially reduced enzyme is a light-activated disproportionation since it is totally dependent upon the presence of some oxidized enzyme. Enzyme fully reduced by dithionite forms no semiquinone, while enzyme partially reduced by dithionite rapidly forms semiquinone upon irradiation. Furthermore, the light-activated disproportionation of enzyme first reduced with NADPH results in the reduction of NADP. Thus, FAD catalyzes the disproportionation in keeping with the known photosensitizing nature of free flavins. This reaction is reversed slowly (half-time ca. 150 min 25°) in the dark. The semiquinone is rapidly reoxidized by oxygen to yield an enzyme with unaltered spectral and catalytic properties (58). Similar reactions have been very briefly reported for lipoamide dehydrogenase the dark reverse reaction is comparatively rapid, being complete in 30 min (16S). 

The oxidant in this reaction is the disulfide group of lipoamide, which is reduced to its disulfhydryl form. This reaction, also catalyzed by the pyruvate dehydrogenase component Ej, yields acetyllipoamide. 

E3 oxidizes the reduced lipoamide arm by transferring two hydrogens to FAD, forming FADH2... 

Fig. 11-15 The pyruvate dehydrogenase reaction takes piace via three enzymes in a complex Pyruvate is decarboxyiated by the pyruvate decarboxylase) component of the enzyme complex a key cofactor is thiamine pyrophosphate (TPP) that transfers the hydroxyethyl moiety to one of the sulfur atoms on oxidized lipoamide that is covalently bound to Ej(c//hyc/ro//poy/ transacetylase). When this transfer takes place, the 2-carbon moiety is oxidized to an acetyl moiety and then the acetyl moiety is transferred to CoA to yield acetyl-CoA which is then released from the active site of Ej. The reduced lipoamide moiety is recycled back to its oxidized form by donating hydrogen atoms to FAD in E3 lipoamide dehydrogenase), and the reaction cycle begins over again.

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