title: Mechanism of 4-HNE mediated inhibition of hDDAH-1: implications in no regulation.
abstract: Nitric oxide synthase is inhibited by NG-methylated derivatives of arginine whose cellular levels are controlled by dimethylarginine dimethylamino-hydrolase (DDAH). DDAH-1 is a Zn(II)-containing enzyme that through hydrolysis of methylated l-arginines regulates the activity of NOS. Herein, we report the kinetic properties of hDDAH-1 and its redox-dependent regulation. Kinetic studies using recombinant enzyme demonstrated Km values of 68.7 and 53.6 microM and Vmax values of 356 and 154 nmols/mg/min for ADMA and L-NMMA, respectively. This enzymatic activity was selective for free ADMA and L-NMMA and was incapable of hydrolyzing peptide incorporated methylarginines. Subsequent studies performed to determine the effects of reactive oxygen and reactive nitrogen species on DDAH activity demonstrated that low level oxidant exposure had little effect on enzyme activity and that concentrations approaching >or=100 microM were needed to confer significant inhibition of DDAH activity. However, exposure of DDAH to the lipid oxidation product, 4-HNE, dose-dependently inhibited DDAH activity with 15% inhibition observed at 10 microM, 50% inhibition at 50 microM, and complete inhibition at 500 microM. Mass spectrometry analysis demonstrated that the mechanism of inhibition resulted from the formation of Michael adducts on His 173, which lies within the active site catalytic triad of hDDAH-1. These studies were performed with pathophysiologicaly relevant concentrations of this lipid peroxidation product and suggest that DDAH activity can be impaired under conditions of increased oxidative stress. Because DDAH is the primary enzyme involved in methylarginine metabolism, the loss of activity of this enzyme would result in impaired NOS activity and reduced NO bioavailability.
authors.affiliation: Department of Physiology and Functional Genomics, The University of Florida College of Medicine, Gainesville, Florida 32610, USA.
authors.forename: Scott P
authors.initials: SP
authors.lastname: Forbes
authors.forename: Lawrence J
authors.initials: LJ
authors.lastname: Druhan
authors.forename: Jorge E
authors.initials: JE
authors.lastname: Guzman
authors.forename: Narasimham
authors.initials: N
authors.lastname: Parinandi
authors.forename: Liwen
authors.initials: L
authors.lastname: Zhang
authors.forename: Kari B
authors.initials: KB
authors.lastname: Green-Church
authors.forename: Arturo J
authors.initials: AJ
authors.lastname: Cardounel
mesh_terms: D000447:Aldehydes / Q000494:pharmacology*; D000581:Amidohydrolases / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000302:isolation & purification; D000595:Amino Acid Sequence; D004591:Electrophoresis, Polyacrylamide Gel; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D016877:Oxidants / Q000494:pharmacology; D020543:Proteome; D053719:Tandem Mass Spectrometry
chemical_list: D000447:Aldehydes; D004791:Enzyme Inhibitors; D016877:Oxidants; D020543:Proteome; D000581:Amidohydrolases; C059999:dimethylargininase; C027576:4-hydroxy-2-nonenal