Differential Effects of Mercurial on the Lipoyl Reductase and Diaphorase Activities of Lipoyl Dehydrogenase

Abstract

Abstract The sensitivity of lipoyl dehydrogenase to phenylmercuric acetate, a reagent for —SH groups, has been investigated. Titration of available sulfhydryl groups in the enzyme brings about inactivation in the reduced diphosphopyridine nucleotide-lipoyl reductase activity, while activity with the artificial hydrogen acceptor 2,6-dichloroindophenol (DCI) is stimulated. Similar results are found upon treating the enzyme with phenylmercuric acetate after prior reduction by DPNH, except that a lower level of mercurial is required for inhibition of oxidized dl-α-lipoic acid activity, and DCI activity is more stable. Upon reduction with DPNH the semiquinone of the mercurial-treated enzyme is initially produced, but is no longer stable; the enzyme has a much greater tendency to be reduced to the 4-electron reduction state. Enzyme allowed to react with phenylmercuric acetate for a long period of time is lacking in DPNH-DCI reductase activity, and shows very slow reduction through the semiquinone when treated with DPNH. Evidence is presented that the effect of mercurial on both DPNH-lipoyl and DPNH-DCI reductase activities is due to transfer of mercurial to the active center during catalysis. A schematic representation of the reaction mechanism is presented which shows the presumed reasons for the changes in the catalytic properties of lipoyl dehydrogenase after mercurial treatment.