Structural analysis of porcine brain nitric oxide synthase reveals a role for tetrahydrobiopterin and L-arginine in the formation of an SDS-resistant dimer.
Nitric oxide synthases (NOSs), which catalyze the formation of the ubiquitous biological messenger molecule nitric oxide, represent unique cytochrome P-450s, containing reductase and mono-oxygenase domains within one polypeptide and requiring tetrahydrobiopterin as cofactor. To investigate whether tetrahydrobiopterin functions as an allosteric effector of NOS, we have analyzed the effect of the pteridine on the conformation of neuronal NOS purified from porcine brain by means of circular dichroism, velocity sedimentation, dynamic light scattering and SDS-polyacrylamide gel electrophoresis. We report for the first time the secondary structure of NOS, showing that the neuronal isozyme contains 30% alpha-helix, 14% antiparallel beta-sheet, 7% parallel beta-sheet, 19% turns and 31% other structures. The secondary structure of neuronal NOS was neither modulated nor stabilized by tetrahydrobiopterin, and the pteridine did not affect the quaternary structure of the protein, which appears to be an elongated homodimer with an axial ratio of approximately 20/1 under native conditions. Low temperature SDS-polyacrylamide gel electrophoresis revealed that tetrahydrobiopterin and L-arginine synergistically convert neuronal NOS into an exceptionally stable, non-covalently linked homodimer surviving in 2% SDS and 5% 2-mercaptoethanol. Ligand-induced formation of an SDS-resistant dimer is unprecedented and suggests a novel role for tetrahydrobiopterin and L-arginine in the allosteric regulation of protein subunit interactions.
Klatt
P
P
Institut für Pharmakologie und Toxikologie, Karl-Franzens-Universität Graz, Austria.
Schmidt
K
K
Lehner
D
D
Glatter
O
O
Bächinger
H P
HP
Mayer
B
B
D000494:Allosteric Regulation; D000594:Amino Acid Oxidoreductases / Q000737:chemistry* / Q000302:isolation & purification; D000818:Animals; D001120:Arginine / Q000494:pharmacology*; D001708:Biopterins / Q000031:analogs & derivatives* / Q000494:pharmacology; D001923:Brain Chemistry; D006358:Hot Temperature; D009474:Neurons / Q000201:enzymology; D019001:Nitric Oxide Synthase; D011487:Protein Conformation / Q000187:drug effects*; D017510:Protein Folding; D017433:Protein Structure, Secondary; D012967:Sodium Dodecyl Sulfate; D013552:Swine
D001708:Biopterins; D012967:Sodium Dodecyl Sulfate; D001120:Arginine; D019001:Nitric Oxide Synthase; D000594:Amino Acid Oxidoreductases; C003402:sapropterin