title: Anti-pterins as tools to characterize the function of tetrahydrobiopterin in NO synthase.
abstract: Nitric oxide synthases (NOS) are homodimeric enzymes that NADPH-dependently convert L-arginine to nitric oxide and L-citrulline. Interestingly, all NOS also require (6R)-5,6,7, 8-tetrahydro-L-biopterin (H4Bip) for maximal activity although the mechanism is not fully understood. Basal NOS activity, i.e. that in the absence of exogenous H4Bip, has been attributed to enzyme-associated H4Bip. To elucidate further H4Bip function in purified NOS, we developed two types of pterin-based NOS inhibitors, termed anti-pterins. In contrast to type II anti-pterins, type I anti-pterins specifically displaced enzyme-associated H4Bip and inhibited H4Bip-stimulated NOS activity in a fully competitive manner but, surprisingly, had no effect on basal NOS activity. Moreover, for a number of different NOS preparations basal activity (percent of Vmax) was frequently higher than the percentage of pterin saturation and was not affected by preincubation of enzyme with H4Bip. Thus, basal NOS activity appeared to be independent of enzyme-associated H4Bip. The lack of intrinsic 4a-pterincarbinolamine dehydratase activity argued against classical H4Bip redox cycling in NOS. Rather, H4Bip was required for both maximal activity and stability of NOS by binding to the oxygenase/dimerization domain and preventing monomerization and inactivation during L-arginine turnover. Since anti-pterins were also effective in intact cells, they may become useful in modulating states of pathologically high nitric oxide formation.
authors.affiliation: Department of Pharmacology and Toxicology, Julius-Maximilians-University, D-97078 Würzburg, Germany.
authors.forename: H M
authors.initials: HM
authors.lastname: Bömmel
authors.forename: A
authors.initials: A
authors.lastname: Reif
authors.forename: L G
authors.initials: LG
authors.lastname: Fröhlich
authors.forename: A
authors.initials: A
authors.lastname: Frey
authors.forename: H
authors.initials: H
authors.lastname: Hofmann
authors.forename: D M
authors.initials: DM
authors.lastname: Marecak
authors.forename: V
authors.initials: V
authors.lastname: Groehn
authors.forename: P
authors.initials: P
authors.lastname: Kotsonis
authors.forename: M
authors.initials: M
authors.lastname: La
authors.forename: S
authors.initials: S
authors.lastname: Köster
authors.forename: M
authors.initials: M
authors.lastname: Meinecke
authors.forename: M
authors.initials: M
authors.lastname: Bernhardt
authors.forename: M
authors.initials: M
authors.lastname: Weeger
authors.forename: S
authors.initials: S
authors.lastname: Ghisla
authors.forename: G D
authors.initials: GD
authors.lastname: Prestwich
authors.forename: W
authors.initials: W
authors.lastname: Pfleiderer
authors.forename: H H
authors.initials: HH
authors.lastname: Schmidt
mesh_terms: D000818:Animals; D001708:Biopterins / Q000031:analogs & derivatives* / Q000037:antagonists & inhibitors / Q000378:metabolism; D002384:Catalysis; D002531:Cerebellum / Q000201:enzymology; D006801:Humans; D007700:Kinetics; D019001:Nitric Oxide Synthase / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D013552:Swine
chemical_list: D011994:Recombinant Proteins; D001708:Biopterins; D019001:Nitric Oxide Synthase; C003402:sapropterin