title: Inactivation of porcine kidney betaine aldehyde dehydrogenase by hydrogen peroxide.
abstract: Concentrated urine formation in the kidney is accompanied by conditions that favor the accumulation of reactive oxygen species (ROS). Under hyperosmotic conditions, medulla cells accumulate glycine betaine, which is an osmolyte synthesized by betaine aldehyde dehydrogenase (BADH, EC 1.2.1.8). All BADHs identified to date have a highly reactive cysteine residue at the active site, and this cysteine is susceptible to oxidation by hydrogen peroxide. Porcine kidney BADH incubated with H(2)O(2) (0-500 μM) lost 25% of its activity. However, pkBADH inactivation by hydrogen peroxide was limited, even after 120 min of incubation. The presence of coenzyme NAD(+) (10-50 μM) increased the extent of inactivation (60%) at 120 min of reaction, but the ligands betaine aldehyde (50 and 500 μM) and glycine betaine (100 mM) did not change the rate or extent of inactivation as compared to the reaction without ligand. 2-Mercaptoethanol and dithiothreitol, but not reduced glutathione, were able to restore enzyme activity. Mass spectrometry analysis of hydrogen peroxide inactivated BADH revealed oxidation of M278, M243, M241 and H335 in the absence and oxidation of M94, M327 and M278 in the presence of NAD(+). Molecular modeling of BADH revealed that the oxidized methionine and histidine residues are near the NAD(+) binding site. In the presence of the coenzyme, these oxidized residues are proximal to the betaine aldehyde binding site. None of the oxidized amino acid residues participates directly in catalysis. We suggest that pkBADH inactivation by hydrogen peroxide occurs via disulfide bond formation between vicinal catalytic cysteines (C288 and C289).
authors.affiliation: Coordinación de Ciencia de los Alimentos, Centro de Investigación en Alimentación y Desarrollo AC, Sonora, Mexico.
authors.forename: Jesús A
authors.initials: JA
authors.lastname: Rosas-Rodríguez
authors.forename: Elisa M
authors.initials: EM
authors.lastname: Valenzuela-Soto
mesh_terms: D000818:Animals; D050728:Betaine-Aldehyde Dehydrogenase / Q000737:chemistry / Q000378:metabolism*; D004789:Enzyme Activation / Q000187:drug effects; D004793:Enzyme Reactivators / Q000494:pharmacology; D004795:Enzyme Stability / Q000187:drug effects; D006861:Hydrogen Peroxide / Q000494:pharmacology*; D007668:Kidney / Q000201:enzymology*; D007700:Kinetics; D008024:Ligands; D008958:Models, Molecular; D011487:Protein Conformation; D013552:Swine
chemical_list: D004793:Enzyme Reactivators; D008024:Ligands; D006861:Hydrogen Peroxide; D050728:Betaine-Aldehyde Dehydrogenase