Studies on the Mechanism of Action of Acetyl Coenzyme A Carboxylase II. ON THE MECHANISM OF ACTION OF ENZYME-BOUND BIOTIN” MOSELEY

Ever since the identification of biotin with the curative factor (vitamin H) for egg white injury (1, 2), investigators have concerned themselves with the mode and mechanism of action of this vitamin. With the use of both microbiological techniques and animal experiments, early investigators recognized various biochemical roles for biotin, especially as a key component in the metabolism of COZ, fatty acids, and various dicarboxylic acids. More specifically, biotin was shown somehow to be required for various carboxylation reactions, including the synthesis of oxaloacetic acid from pyruvate plus COz (3-7). The sparing effect of oleic acid on biotin requirements suggested that the latter may play a role in the synthesis of fatty acids (8-11). Biotin deficiency in animals was also known to cause a reduction in the levels of malic enzyme, citrulline synthetase, and other enzymes (12-19). Melville, Pierce, and Partridge (20) first postulated that biotin may function in carboxylation reactions through the turnover of the carbonyl group of the ureido ring. These workers synthesized ureido-U4-biotin and used it as a growth factor for Lactobacillus arabinosus. Biotin was reisolated from both cells and media and its Cl4 content was determined. Approximately 93 y. of the initial Cl4 was still associated with the biotin; this high recovery seemed to invalidate the aforementioned hypothesis, since if the carbonyl carbon of biotin were the group involved in carboxylation reactions, they would have found little or no radioactivity in biotin. In 1958 Wakil, Titchener, and Gibson (21) and Wakil and Gibson (22) observed that bicarbonate was required for fatty acid synthesis and that one of the enzymes involved in fatty acid synthesis contained large amounts of biotin. Biotin was tightly bound to the protein and was closely associated with the enzymatic activity throughout the purification steps. In addition, Wakil and Gibson (22) demonstrated that avidin, an egg white protein long known for its high binding affinity for biotin (23), inhibited the carboxylation of acetyl-CoA. Treatment of avidin with free biotin, before its interaction with the enzyme, prevented