Lipoic acid proteins, attachment

Biotin (vitamin H) is an unusual vitamin because it is synthesized by bacteria that live in the intestine. Consequently, biotin does not have to be included in our diet, and deficiencies are rare. Biotin deficiencies, however, can be found in people who maintain a diet high in raw eggs. Egg whites contain a protein (called avidin) that binds biotin tightly and thereby prevents it from acting as a coenzyme. When eggs are cooked, avidin is denatured, and the denatured protein does not bind biotin. Biotin, like lipoic acid, is attached to its enzyme (E) by forming an amide with the amino group of a lysine side chain. 

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). 

The -amino groups of lysyl residues serve as attachment sites of a number of coenzymes in proteins (e.g. biotin in pyruvate carboxylase, pyridoxal phosphate in phosphorylase, lipoic acid in lipoate acetyl-transferase) and form covalent intermediates in several enzymic reactions (e.g. transaldolase, aldolase, etc.). Discussion of all of these naturally-occuring derivatives of lysine will not be attempted in this treatise, but the investigator using chemical modification of proteins should be aware of their possible presence and effect on the results of his experiments. It should be noted that e-N-phospholysine has been reported in nucleoside diphosphate kinase (Walinder 1968). 

In neither of the above studies was the isolated protein shown to catalyze the formation of lipoic add. Indeed, at this time, confusion about the identity of the substrate for the reaction was a major issue. Octanoic acid was initially hypothesized to be the substrate, because experimental evidence indicated that it is converted intact into lipoic acid in vivo. However, null mutations in the IplA gene, which encodes the protein that would catalyze attachment of lipoic acid to LCPs if it were synthesized from octanoic acid, did not result in growth defects unless either the lipA or lipB genes also contained null mutations, indicating a second, redundant, pathway for biosynthesis of the lipoyl cofactor in E. coli. Because octanoic acid was known to be synthesized de novo by type II fatty acid biosynthesis, it followed that the substrate might be an intermediate in the fatty acid biosynthetic pathway. Since octanoyl-E2 domains had been shown not to be converted into lipoyl—E2 domains, it was assumed that the substrate for LipA was octanoyl-ACP, and that the substrate for LipB was lipoyl—AGP rather than octanoyl—AGP. 

Figure 16 Proposed mechanism of the LipA reaction. Cataiysis occurs on an octanoyi substrate attached in an amide linkage to a lipoyl carrier protein (LCP). In the first half-reaction, reductive cleavage of SAM to generate a 5 -deoxyadenosyl 5 -radical allows for hydrogen atom abstraction at C6 of the fatty acyl chain. The proposed source of sulfur is a second [4Fe-4S] cluster, housed in the CX4CX5C motif conserved in lipoyl synthases. In the second half-reaction, the second 5 -deoxyadenosyl 5 -radical abstracts a hydrogen atom from C8 of the fatty acyl chain, allowing for sulfur insertion at this position. The addition of two protons affords lipoic acid in its reduced form.

Lipoamide Cofactor form of oxidized (cyclic disulfide) lipoic acid, in which it is attached covalently to the e-amino group of a target Iysyi residue on a lipoyl carrier protein. 

Lipoyl carrier protein A protein to which lipoic acid is covalently attached in an amide linkage to the e nitrogen atom of a specific Iysyi residue. LCPs employ the lipoyl group as a cofactor in enzymatic catalysis. 

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. 

FIGURE 11 The vitamin biotin and the vitamin-like compound iipoic acid and their covaient attachments to selected lysine side chains in proteins (poiypeptides). Both of these compounds function as cataiytic prosthetic groups, biotin for CO2 and lipoic acid for hydrogen. The fragment biocytin was isoiated from autolysates of rapidly growing yeast. 

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