Lipoic acid (1,2-dithiolane-3-pentanoic acid or, trivially, thioctic acid) is chiefly known as a protein-bound cofactor in the oxidative decarboxylation of 2-oxo acids. 2-Oxo acid dehydrogenase multienzyme complexes catalyze the oxidative decarboxylation of pyruvate, 2-oxoglutarate, and the branched chain 2-oxo acids that are derived from the transamination of leucine, isoleucine, and valine. In all these instances, the lipoyl group is found in amide linkage with the N6-amino group of a specific lysine residue in the dihydrolipoyl acyltransferase (E2) component, where it acts as a "swinging arm" to ferry the substrate between the three active sites that successively catalyze the overall reaction (1-3). A similar lipoyl-lysine residue occurs in another widespread multienzyme system, the glycine cleavage system, which catalyzes the decarboxylation of glycine (4, 5). All lipoylated protein structures studied thus far contain an autonomously folded domain of about 80 amino acid residues, in which the lipoyl-lysine residue is displayed in a prominent b-turn (6-9). The selectivity of the lipoyl protein ligase that catalyzes the lipoylation reaction (10, 11) is unusual, depending in large part on the correct siting of the target lysine residue in the exposed b-turn of the apo-lipoyl domain (12). Free lipoic acid is inactive as a substrate in the 2-oxo acid dehydrogenase complexes and must be attached to the lipoyl domain to play its part in the systems of active-site coupling and substrate channeling that are prominent features of these complexes (13). There are strong parallels (13) between these properties of lipoic acid and those of biotin in various ATP-dependent carboxylases and 4-phosphopantothenic acid in fatty acid and polyketide synthases (see Thiotemplate Mechanism Of Peptide Antibiotic Synthesis). Lipoic acid has additionally been attributed to numerous other biological effects, among them that of a protective antioxidant against free radicals, an inhibitor of lipid peroxidation and of HIV replication, and a valuable dietary supplement (14, 15).
1. Structure and Biosynthesis
Lipoic acid was first discovered in the quest for the "pyruvate oxidation factor" (16). It is based on the eight-carbon fatty acid, octanoic acid, modified by the insertion of sulfur atoms at C-6 and C-8 to give a dithiolane ring (Fig. 1). There is a chiral carbon atom at position 6, and generally only the R-enantiomer is active in the 2-oxo acid dehydrogenase complexes (17). There are intriguing similarities between the enzymes, which contain [2Fe-2 S] clusters (see Iron-Sulfur Proteins), responsible for the insertion of sulfur into the biotin and lipoic acid precursors (18).
2. The Lipoyl Domain
The E2 polypeptide chain of the 2-oxo acid dehydrogenase complexes consists of, from the N- terminus: one to three lipoyl domains (depending on the source), a peripheral subunit-binding domain, and a catalytic (acyltransferase) domain. The domains are joined together by long (20 to 30 residue) and conformationally flexible linker regions, and the catalytic domain aggregates with octahedral (24-mer) or icosahedral (60-mer) symmetry (again according to source), to form the structural core of the complex (1-3). Lipoic acid will not serve as a substrate for the 2-oxo acid decarboxylase (E1) component unless attached to the lipoyl domain (the value of kcat/Km is thereby raised by a factor of 104); moreover, the lipoyl domain restricts reductive acylation of the lipoyl group to the partner E1 of the parent complex. Thus the true substrate is the conformationally mobile lipoyl domain, an elegant molecular basis for substrate channeling and active-site coupling in these complexes (2, 13).
Figure 1. Structure of lipoic acid (a) and its biosynthetic precursor, octanoic acid (b).
The structures of several lipoyl domains have been determined by means of nuclear magnetic resonance spectroscopy (6-8). All consist of two four-stranded b-sheets, with the lipoyl-lysine residue displayed in a prominent b-turn in one sheet, and the N- and C-termini close together in space in the other sheet, related by a two-fold axis of quasi-symmetry (Fig. 2). The structure of the lipoylated H-protein of the glycine cleavage system is similar (9). Interaction of the lipoyl domain with E1 is only transient, and the specificity appears to depend in large part on the nature of the amino acid residues surrounding the lipoyl-lysine residue in its b-turn and on the neighboring surface loop between strands 1 and 2 (19). Curiously, in the human autoimmune disease, primary biliary cirrhosis, the offending antigen is the lipoylated domain of the E2 component of the pyruvate dehydrogenase complex (20), which is normally a mitochondrial protein.
Figure 2. Structure of the lipoyl domain of the dihydrolipoyl succinyltransferase component of the 2-oxoglutarate dehydrogenase complex of Escherichia coli (based on Ref. 7). The b-sheet containing the lipoyl-lysine residue is shown in dark shading, and the b-sheet containing the N – and C-terminal residues is shown in light shading. The lipoylation site (Lys43) in the turn between strands 4 and 5 is indicated.
3. Post-translational Modification
The lipoyl group is attached to the target lysine residue in the lipoyl domain by an ATP-dependent enzyme, lipoyl protein ligase (10, 11). Its two-step mechanism resembles that of a fatty acyl CoA synthetase or aminoacyl tRNA synthetase (Fig. 3). The specificity of the post-translational modification depends crucially on the correct siting of the target lysine residue in the exposed b-turn of the apo-lipoyl domain, and much less on the surrounding amino acid sequence (12). In this it differs significantly from many other post-translational modifications, for which the sequence motif is dominant.
Figure 3. Reactions catalyzed by biotinyl and lipoyl protein ligases.
4. Similarities with Biotin
Biotin-lysine is the swinging arm carrying the carboxy group in multienzyme systems that catalyze carboxylation reactions. Like lipoic acid, it too is attached in amide linkage to the N 6-amino group of a specific lysine residue in the relevant enzyme. There are intriguing similarities between biotin and lipoic acid: (a) in the enzymes that catalyze their biosynthesis; (b) in the ability of both to bind tightly to avidin (although lipoic acid does so with a much higher dissociation constant, ~10–6M); (c) in the existence of a biotinyl domain in biotin-dependent enzymes, the structure of which closely resembles that of the lipoyl domain (13, 21); (d) in the requirement for the lipoic acid or biotin to be attached to the lipoyl or biotinyl domain before it will serve as a substrate in its parent enzyme complex; (e) in the biotinylation of the target lysine residue catalyzed by an enzyme that mechanistically resembles lipoyl protein ligase (Fig. 3); and (f) in the dependence of posttranslational modification on the siting of the target lysine residue in the exposed b-turn of the apodomain(13).
5. Role as a Swinging Arm
There is clear evidence for the essentially free rotation of the swinging arm on the surface of the lipoyl domain of 2-oxo acid dehydrogenase complexes (2, 3). However, the lipoyl-lysine residue in the H-protein of the glycine cleavage system is localized by interactions with the protein (9). It switches to a new position when charged with substrate, such that the aminomethylated derivative is sequestered in a surface cavity of the domain unique to the H-protein (9). In this instance, the swinging arm is fulfilling the expectation of the “hot potato hypothesis” of multienzyme complexes,
protecting an unstable intermediate for presentation to the next enzyme in the sequence (13).Likewise, the biotinyl-lysine residue of the biotinyl domain of the acetyl CoA carboxylase of E. coli is clearly localized by interaction with the protein (21), but there is no evidence of similar biotin–protein interactions in the 1.3 S subunit of Propionibacterium shermanii transcarboxylase (22). It is not known what purpose, if any, is served by the prior localization of the biotinyl lysine residue in biotin-dependent reactions.
