Abstract
Studies on the activation of an apopyruvate dehydrogenation system obtained from extracts of lipoic acid-deficient Streptococcus jaecalis cells indicated that in its functional form lipoic acid is bound to protein in covalent linkage through its carboxyl group, i.e. as “lipoyl enzyme” (1). Further support for this proposal is furnished by the present finding that a partially purified enzyme, “lipoyl-X hydrolase,” obtained from S. juecalis extracts, liberated lipoic acid from the protein-bound form present in the Escherichia coli (Crookes strain) pyruvate dehydrogenation system, thereby inactivating the latter system. Reactivation required the same components and conditions as were found necessary to activate the S. jaecalis apopyruvate dehydrogenation system. A preliminary communication of this work has been reported elsewhere (2). The availability of a method of releasing lipoic acid from the proteinbound form and of reactivating the apoenzyme has enabled us to study the mechanism of certain enzymatic reactions which have been carried out with free lipoic acid or structurally related compounds. Gunsalus and his collaborators reported (3-6) that E. coli Fraction A, in the presence of CoA’ and phosphotransacetylase, catalyzed Reactions 1 and 2, and that Fraction B, in the presence of DPN and lactic dehydrogenase, catalyzed Reaction 3.
Highlights
Activity of Lipoamides with Enzyme Preparations from S. faecalis-During the course of studies with cell-free extracts of lipoic acid-deficient S. faecalis, LEVITCH, several amides of lipoic acid were synthesized for testing as antagonists of lipoic acid
Participation of Protein-Bound Lipoic Acid in Model Reactions-Evidence pertaining to mechanisms (b) and (c) was obtained by measuring the enzymatic activities, as exhibited in Reactions 1,2, and 3, of pyruvate and apopyruvate dehydrogenation systems prepared from X. faecalis and E. coli
An enzyme preparation from lipoic acid-deficient Streptococcus faecalis is described which released lipoic acid from the protein-bound form present in pyruvate dehydrogenation systems prepared from Escherichia coli and S. faecalis, yielding apopyruvate dehydrogenation systems
Summary
Reaction 6, with free lipoic acid or lipoamide as substrate, is catalyzed by a purified mammalian cw-ketoglutarate dehydrogenation complex [7]. Since Fraction A and the a-ketoglutarate dehydrogenation complex contain protein-bound lipoic acid [5, 9], which presumably functions catalytically in Reaction 8, it has not been possible to decide whether the reactions which occur with free lipoic acid, dihydrolipoic acid, or the corresponding amides involve (a) exchange between free and protein-bound lipoic acid; (b) coupling between free and protein-bound lipoic acid; or (c) reaction of the appropriate enzyme with free material. A basis for deciding which of these mechanisms is applicable to Reactions 1, 5, and 6 is provided in the present communication
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