Glutathionylation of α-ketoglutarate dehydrogenase: The chemical nature and relative susceptibility of the cofactor lipoic acid to modification

The mitochondrial enzyme α-ketoglutarate dehydrogenase (KGDH)1 is a large multienzyme complex that consists of multiple copies of three subunits: E1 (α-ketoglutarate dehydrogenase), E2 (dihydrolipoyl succinyltransferase), and E3 (dihydrolipoyl dehydrogenase). Together, these subunits catalyze the exergonic \conversion of α-ketoglutarate, CoASH, and NAD+ to CO2, succinyl-CoA, and NADH. Enzyme activity is stimulated by Ca2+ and inhibited by the catalytic products succinyl-CoA and NADH to reflect metabolic demand [1–9]. As a key regulatory site within the Krebs cycle, KGDH plays a critical role in cellular energy and biosynthetic homeostasis [10–14]. It is therefore noteworthy that loss of KGDH activity has been observed during numerous degenerative diseases [14–22]. Many of these diseases are associated with oxidative stress. In vitro, KGDH has been shown to be a sensitive target of oxidative damage [22–25]. Oxidative inactivation of KGDH is therefore thought to contribute to the cellular and tissue dysfunction characteristic of these pathophysiological conditions. In sharp contrast to oxidative damage, KGDH can also undergo reversible inhibition in response to oxidative stress. Treatment of isolated rat cardiac mitochondria with H2O2 results in a loss of KGDH activity and in the rate of ADP-dependent mitochondrial respiration. Upon consumption of H2O2, KGDH and respiratory activities are restored [26]. H2O2-induced inhibition of KGDH is due to formation of a mixed disulfide between a protein sulfhydryl and glutathione (glutathionylation) [27]. In vitro, glutathionylation protects catalytic sulfhydryls on KGDH from oxidative damage [28]. Thus, the reversible nature of this modification could provide for redox regulation as a means to protect the enzyme from oxidative damage. If so, why then would KGDH undergo irreversible oxidative inactivation during certain pathophysiological processes? To adequately address this question, the mechanisms by which KGDH is glutathionylated and the physiological significance of this form of enzyme regulation must be established. The transient nature of glutathionylation coupled with difficulties in detection has limited mechanistic investigations. Additionally, in disrupted mitochondria or in purified form, KGDH is resistant to glutathionylation-dependent inhibition that can be reversed by glutaredoxin [27]. Previous studies that took advantage of chemical derivatization and sulfhydryl reactivity indirectly identified lipoic acid, a required cofactor covalently linked to the E2 subunit, as the likely site of glutathionylation [28]. This result is, however, perplexing because of the chemical properties of lipoic acid. Lipoic acid has two vicinal sulfhydryls. Therefore, rapid disulfide exchange upon glutathionylation would be predicted to render lipoic acid resistant to stable modification. It is also not clear why H2O2-induced inhibition of KGDH does not exceed 50% [26–28]. This study sought conclusive identification of the site and chemistry of KGDH glutathionylation and factors that control the degree and rate of enzyme inhibition. Chemical, biochemical, and immunochemical approaches were utilized. The results offer important insights on the properties of KGDH that make lipoic acid susceptible to stable glutathionylation and establish a reliable means for exploring the mechanism by which this process occurs.