A Trail of Research from Lipoic Acid to α-Keto Acid Dehydrogenase Complexes

Abstract

pyruvate dehydrogenase α-ketoglutarate dehydrogenase binding protein truncated E2 In this article I shall retrace a trail of research that began with the isolation and characterization of a microbial growth factor and led to elucidation of the structure, function, and regulation of α-keto acid dehydrogenase complexes. The high points of this trail are presented below. This trail of discovery started in the spring of 1949, about 6 months after I joined the faculty of the Department of Chemistry at the University of Texas. At that time I started working on the isolation of a factor that replaced acetate in the growth medium for certain lactic acid bacteria. Research on the “acetate-replacing factor” was initiated by Esmond Snell and associates at the University of Wisconsin and then at the University of Texas. I inherited this project in the spring of 1949. We established that this factor is widely distributed in animal, plant, and microbial cells and that liver is a rich source. The factor is tightly bound to liver protein and is released by proteolysis or by acid hydrolysis. At that time pharmaceutical companies were processing large amounts of pork and beef liver to obtain extracts suitable for treatment of pernicious anemia. The active principle was shown later to be vitamin B12. Fresh liver was extracted with warm water, and the residual liver proteins and fatty material were dried and sold as an animal feed supplement. Arrangements were made with Eli Lilly and Co. to obtain liver residue, and we developed procedures for extracting and purifying the acetate-replacing factor. We progressed to the point of being able to process about 6 pounds of liver residue at a time. A 16,000–50,000-fold purification was achieved. In the late 1940s and early 1950s several other groups were trying to isolate factors that were similar to, if not identical with, the acetate-replacing factor. These factors included the “pyruvate oxidation factor” of I. C. Gunsalus and associates that was necessary for oxidation of pyruvate to acetate and carbon dioxide byStreptococcus faecalis cells grown in a synthetic medium. Gunsalus was also collaborating with Eli Lilly and Co. In the fall of 1950, the Lilly Research Laboratories merged the two separate collaborations to facilitate isolation of the acetate-replacing/pyruvate oxidation factor. The Lilly group adapted and scaled up isolation procedures developed by us. Instead of processing 6-pound batches of liver residue at a time, they were able (using commercial equipment) to process 250-pound batches. Concentrates of the factor that were 0.1–1% pure were sent to my laboratory for further processing. I obtained the first pale yellow crystals of the factor, about 3 mg, on or about March 15, 1951, a truly memorable occasion. It was partially characterized and given the trivial name α-lipoic acid (1Reed L.J. DeBusk B.G. Gunsalus I.C. Hornberger Jr., C.S. Crystalline α-lipoic acid: a catalytic agent associated with pyruvate dehydrogenase..Science. 1951; 114: 93-94Crossref PubMed Scopus (224) Google Scholar). The isolation involved a 300,000-fold purification. A total of ∼30 mg of crystalline lipoic acid was eventually isolated. We estimated that ∼10 tons of liver residue were processed to obtain this small amount of the pure substance. And to think that I was processing about 6 pounds of liver residue at a time, convinced that I would eventually isolate the pure material. We established that lipoic acid is a cyclic disulfide, either 6,8-, 5,8-, or 4,8-dithiooctanoic acid. That the correct structure is 6,8-dithiooctanoic acid (1,2-dithiolane-3-valeric acid) was established by synthesis of dl-lipoic acid, first achieved by E. L. R. Stokstad and associates at Lederle Laboratories. I was intrigued by this simple yet unique substance and wanted to know more about its biological function, i.e. with what and how does it function in living cells. Little did I know then that this trail would take me through five decades of research that has turned out to be a fascinating and rewarding chapter of modern biochemistry, elucidation of the mechanism of oxidative decarboxylation of α-keto acids. Prior to the isolation of lipoic acid and its characterization as a cyclic disulfide, contributions from several laboratories had established the cofactor requirements for the oxidative decarboxylation of α-keto acids represented by the equation shown below. RCOCO2−+CoASH+NAD+→RCOSCoA+CO2+NADHIn addition to CoA and NAD+, thiamin diphosphate, a divalent metal ion, and protein-bound lipoic acid are required. A requirement for FAD was demonstrated later. The presence of a disulfide linkage in lipoic acid led Gunsalus to propose that lipoic acid underwent a cycle of reactions in α-keto acid oxidation comprising reductive acylation, acyl transfer, and electron transfer. Lipoic acid was visualized as functioning after thiamin diphosphate and before CoA and NAD+. Gunsalus, Lowell Hager, and associates obtained evidence