title: NAD(H) enhances the Cu(II)-mediated inactivation of lactate dehydrogenase by increasing the accessibility of sulfhydryl groups.
abstract: Copper ions are known to inactivate a variety of enzymes, and lactate dehydrogenase (LDH) is exceptionally sensitive to the presence of this metal. We now found that NADH strongly enhances the Cu(II)-mediated loss of LDH activity. Surprisingly, NADH was not oxidized in this process and also NAD+ promoted the Cu(II)-dependent inactivation of LDH. Catalase only partly protected the enzyme, whereas hypoxia even enhanced LDH inactivation. NAD(H) accelerated sulfhydryl (SH) group oxidation of LDH by 5,5-dithio-bis(2-nitrobenzoic acid) (DTNB), and, vice versa, LDH-mediated Cu(II) reduction. LDH activity was preserved by thiol donators and pyruvate and partially preserved by lactate and oxamate. Our results suggest that reactive oxygen species (ROS) are of minor importance for the inactivation of LDH induced by Cu(II)/NADH. We propose that conformational changes of the enzymes' active sites induced by NAD(H)-binding increase the accessibility of active sites' cysteine residues to Cu(II) thereby accelerating their oxidation and, consequently, loss of catalytic activity.
authors.affiliation: Institut für Physiologische Chemie, Universitäitsklinikum, Hufelandstr. 55, D-45122 Essen, Germany.
authors.forename: K
authors.initials: K
authors.lastname: Pamp
authors.forename: T
authors.initials: T
authors.lastname: Bramey
authors.forename: M
authors.initials: M
authors.lastname: Kirsch
authors.forename: H
authors.initials: H
authors.lastname: De Groot
authors.forename: F
authors.initials: F
authors.lastname: Petrat
mesh_terms: D000818:Animals; D001665:Binding Sites; D002384:Catalysis; D002417:Cattle; D003300:Copper / Q000737:chemistry / Q000378:metabolism*; D003545:Cysteine / Q000737:chemistry; D004228:Dithionitrobenzoic Acid / Q000737:chemistry; D004305:Dose-Response Relationship, Drug; D006861:Hydrogen Peroxide / Q000494:pharmacology; D007501:Iron / Q000494:pharmacology; D007770:L-Lactate Dehydrogenase / Q000737:chemistry / Q000378:metabolism*; D007773:Lactates / Q000378:metabolism; D008956:Models, Chemical; D009243:NAD / Q000378:metabolism*; D009711:Nucleotides / Q000737:chemistry; D010084:Oxidation-Reduction; D010100:Oxygen / Q000737:chemistry; D011485:Protein Binding; D011487:Protein Conformation; D011725:Pyridines / Q000737:chemistry; D017382:Reactive Oxygen Species; D013455:Sulfur / Q000737:chemistry*; D013552:Swine; D013997:Time Factors
chemical_list: C045076:Fenton's reagent; D007773:Lactates; D009711:Nucleotides; D011725:Pyridines; D017382:Reactive Oxygen Species; D009243:NAD; D013455:Sulfur; D003300:Copper; D004228:Dithionitrobenzoic Acid; D006861:Hydrogen Peroxide; D007501:Iron; D007770:L-Lactate Dehydrogenase; D003545:Cysteine; C023666:pyridine; D010100:Oxygen