Lipoic acid protein-bound

What structural features of biotin and lipoic acid allow these cofactors to be covalently bound to a specific protein in a multienzyme complex yet participate in reactions at active sites on other enzymes of the complex ... 

E. coli (107, 125). The complexes have recently been reviewed (126). It is possible that lipoamide dehydrogenase also functions in the complexes that oxidatively decarboxylate the a-keto acids resulting from the transamination of valine, isoleucine, and leucine but these have proved difficult to resolve (127). Lipoamide dehydrogenase also functions in the pyridoxal phosphate and tetrahydrofolate-dependent oxidative decarboxylation of glycine in the anaerobic bacterium Peptococcus glyci-nophilus. The reaction in which the protein-bound lipoic acid is reduced is very complex and not yet fully understood the ultimate electron acceptor is NAD+ (112,113,128). 

Figure 1 Formation of protein-bound persulfide and its delivery to sulphur-containing natural compounds.1, cysteine 2, persulfide of a protein bound cysteine 3, rhombic [2Fe-2S] cluster 4, [3Fe-4S] cluster 5, [4Fe-4S] cluster 6, thiamine , lipoic acid 8, molybtopterin 9, biotin.

Biotin (6, Fig. 10) and lipoic acid (7, Fig. 1) are attached enzymatically to apoenzymes via carboxamide linkage to specific lysine residues (60, 61). The pantothenyl moiety (64, Fig. 7) can also be linked covalently to proteins via amide linkage (62). Covalently bound heme is involved in heme M (63) and heme L-catalyzed reactions (64, 65). 

This is an example of several enzymes in which an essential co-factor is covalently bound to the protein. For example, lipoic acid and biotin are covalently linked to the c-amino group of a specific lysine residue in certain enzymes. In some cases, pyridoxal phosphate is bound to the protein through the formation of a Schiff base involving the carbonyl group of the co-factor and an c-amino group of a lysine residue. In cytochrome c, the heme is attached through two thiol ether linkages to cysteine residues of the protein. 

The acyl-generation reaction, Eq. (8), has been visualized as a reductive acylation of protein-bound lipoic acid. As will be seen below, this reaction is now belitwod to consist of two steps an oxidation of the 2-hydroxyalkyl-thiamine pyrophcjsphatc to 2-aoylthiaminc pyrophosphate with a concomitant reduction of bound lipoic acid, and a transfer of (he acyl group of 2-acylthiamine pyrophosphate to the bound dihydrolipoic acid (Das el al., 19(il). An enzymatic component which contains bound lipoic acid and apparently catalyzes reactions (8) and (9) has been isolated from the E. mli pyruvate dehydrogenation complex (Koike and Reed, 1961). This component, designated lipoyl-Ea in Fig. 1, has been tentatively named lipoic reductase-transacetylase. 

The studies of Reed and co-workers on the nature of protein-bound lipoic acid and its enzymatic release and reincorporation may be applicable to biotin-containing enzymes. It is pertinent to note that a conjugated form of biotin, biocytin, has been isolated from yeast autolyzate and identified as A -biotinyl-L-lysine (Wright et ah, 19r)2 Peck et al., 1952). Biotin is now known to be the prosthetic group of several carboxylases (see Ochoa and Kaziro, 1961, for a review of these enzymes). Although the nature of the moiety to which biotin is bound has not been established, it seems highly probable that it is the -amino group of a lysine residue. 

Enzyme systems have been found for the formation and hydrolysis of the lipoyl amide linkage at appropriate lysine e-amino groups of enzymes. The lipoic acid is activated by ATP to form a lipoyl adenylate, possibly as an enzyme-bound form, which then transfers the lipoyl group to the protein amino group. 

It should be noted that most enzyme studies concerning this disulphide-dithiol coenzyme have actually been carried out with either free lipoic acid or lipoamide and not a protein-bound cofactor. While this has en a pragmatic necessity, certain reserve should be maintained in extrapolating from such studies to the protein-bound prosthetic group. 

The reduction of cystine glutathione and proteins with polymer-bound lipoic acid can be carried out either by polymer suspended in the reaction mixture or in a column. 

Thiamine is found in nature in several forms—as free thiamine, the monophosphate, TPP, and probably as thiamine bound by an —S—— linkage to protein, and as the disulfide of TPP. The recent report of Reed and DeBusk indicates still another naturally occurring bound form of thiamine, lipothiamine, the amide of thiamine and a-lipoic acid. Many unsolved problems still remain. In the animal, although TPP constitutes 90% of the total thiamine content in some tissues, in others only 80% occurs as this form muscle may contain more than 50% as free thiamine. The total thiamine content of tissues varies from 10 Mg- per gram wet weight for liver to less than 1 Mg- for muscle and brain. 

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