title: Zinc is a potent inhibitor of thiol oxidoreductase activity and stimulates reactive oxygen species production by lipoamide dehydrogenase.
abstract: Submicromolar zinc inhibits alpha-ketoglutarate-dependent mitochondrial respiration. This was attributed to inhibition of the alpha-ketoglutarate dehydrogenase complex (Brown, A. M., Kristal, B. S., Effron, M. S., Shestopalov, A. I., Ullucci, P. A., Sheu, K.-F. R., Blass, J. P., and Cooper, A. J. L. (2000) J. Biol. Chem. 275, 13441-13447). Lipoamide dehydrogenase, a component of the alpha-ketoglutarate dehydrogenase complex and two other mitochondrial complexes, catalyzes the transfer of reducing equivalents from the bound dihydrolipoate of the neighboring dihydrolipoamide acyltransferase subunit to NAD(+). This reversible reaction involves two reaction centers: a thiol pair, which accepts electrons from dihydrolipoate, and a non-covalently bound FAD moiety, which transfers electrons to NAD(+). The lipoamide dehydrogenase reaction catalyzed by the purified pig heart enzyme is strongly inhibited by Zn(2+) (K(i) approximately 0.15 microm) in both directions. Steady-state kinetic studies revealed that Zn(2+) competes with oxidized lipoamide for the two-electron-reduced enzyme. Interaction of Zn(2+) with the two-electron-reduced enzyme was directly detected in anaerobic stopped-flow experiments. Lipoamide dehydrogenase also catalyzes NADH oxidation by oxygen, yielding hydrogen peroxide as the major product and superoxide radical as a minor product. Zn(2+) accelerates the oxidase reaction up to 5-fold with an activation constant of 0.09 +/- 0.02 microm. Activation is a consequence of Zn(2+) binding to the reduced catalytic thiols, which prevents delocalization of the reducing equivalents between catalytic disulfide and FAD. A kinetic scheme that satisfactorily describes the observed effects has been developed and applied to determine a number of enzyme kinetic parameters in the oxidase reaction. The distinct effects of Zn(2+) on different LADH activities represent a novel example of a reversible switch in enzyme specificity that is modulated by metal ion binding. These results suggest that Zn(2+) can interfere with mitochondrial antioxidant production and may also stimulate production of reactive oxygen species by a novel mechanism.
authors.affiliation: Burke Medical Research Institute, Weill Medical College of Cornell University, White Plains, New York 10605, USA.
authors.forename: Irina G
authors.initials: IG
authors.lastname: Gazaryan
authors.forename: Boris F
authors.initials: BF
authors.lastname: Krasnikov
authors.forename: Gillian A
authors.initials: GA
authors.lastname: Ashby
authors.forename: Roger N F
authors.initials: RN
authors.lastname: Thorneley
authors.forename: Bruce S
authors.initials: BS
authors.lastname: Kristal
authors.forename: Abraham M
authors.initials: AM
authors.lastname: Brown
mesh_terms: D000818:Animals; D008058:Dihydrolipoamide Dehydrogenase / Q000378:metabolism*; D004220:Disulfides; D004305:Dose-Response Relationship, Drug; D007700:Kinetics; D008956:Models, Chemical; D009206:Myocardium / Q000201:enzymology; D009243:NAD / Q000378:metabolism; D010100:Oxygen / Q000378:metabolism; D010101:Oxygen Consumption; D011485:Protein Binding; D011490:Protein Disulfide Reductase (Glutathione) / Q000737:chemistry* / Q000378:metabolism*; D017382:Reactive Oxygen Species; D013379:Substrate Specificity; D013552:Swine; D013997:Time Factors; D015032:Zinc / Q000494:pharmacology* / Q000502:physiology*
chemical_list: D004220:Disulfides; D017382:Reactive Oxygen Species; D009243:NAD; D008058:Dihydrolipoamide Dehydrogenase; D011490:Protein Disulfide Reductase (Glutathione); D015032:Zinc; D010100:Oxygen