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. 1998 May 15;332(Pt 1):195–201. doi: 10.1042/bj3320195

Nitroarginine and tetrahydrobiopterin binding to the haem domain of neuronal nitric oxide synthase using a scintillation proximity assay.

W K Alderton 1, A Boyhan 1, P N Lowe 1
PMCID: PMC1219468  PMID: 9576868

Abstract

Nitric oxide synthases (NOS) have a bidomain structure comprised of an N-terminal oxygenase domain and a C-terminal reductase domain. The oxygenase domain binds haem, (6R)-5,6,7,8-tetrahydro-l-biopterin (tetrahydrobiopterin) and arginine, is the site where nitric oxide synthesis takes place and contains determinants for dimeric interactions. A novel scintillation proximity assay has been established for equilibrium and kinetic measurements of substrate, inhibitor and cofactor binding to a recombinant N-terminal haem-binding domain of rat neuronal NOS (nNOS). Apparent Kd values for nNOS haem-domain-binding of arginine and Nomega-nitro-L-arginine (nitroarginine) were measured as 1.6 microM and 25 nM respectively. The kinetics of [3H]nitroarginine binding and dissociation yielded an association rate constant of 1.3x10(4) s-1.M-1 and a dissociation rate constant of 1.2x10(-4) s-1. These values are comparable to literature values obtained for full-length nNOS, suggesting that many characteristics of the arginine binding site of NOS are conserved in the haem-binding domain. Additionally, apparent Kd values were compared and were found to be similar for the inhibitors, L-NG-monomethylarginine, S-ethylisothiourea, N-iminoethyl-L-ornithine, imidazole, 7-nitroindazole and 1400W (N-[3-(aminomethyl) benzyl] acetamidine). [3H]Tetrahydrobiopterin bound to the nNOS haem domain with an apparent Kd of 20 nM. Binding was inhibited by 7-nitroindazole and stimulated by S-ethylisothiourea. The kinetics of interaction with tetrahydrobiopterin were complex, showing a triphasic binding process and a single off rate. An alternating catalytic site mechanism for NOS is proposed.

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Selected References

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  1. Abrahams J. P., Buchanan S. K., Van Raaij M. J., Fearnley I. M., Leslie A. G., Walker J. E. The structure of bovine F1-ATPase complexed with the peptide antibiotic efrapeptin. Proc Natl Acad Sci U S A. 1996 Sep 3;93(18):9420–9424. doi: 10.1073/pnas.93.18.9420. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Abrahams J. P., Leslie A. G., Lutter R., Walker J. E. Structure at 2.8 A resolution of F1-ATPase from bovine heart mitochondria. Nature. 1994 Aug 25;370(6491):621–628. doi: 10.1038/370621a0. [DOI] [PubMed] [Google Scholar]
  3. Abu-Soud H. M., Yoho L. L., Stuehr D. J. Calmodulin controls neuronal nitric-oxide synthase by a dual mechanism. Activation of intra- and interdomain electron transfer. J Biol Chem. 1994 Dec 23;269(51):32047–32050. [PubMed] [Google Scholar]
  4. Boyhan A., Smith D., Charles I. G., Saqi M., Lowe P. N. Delineation of the arginine- and tetrahydrobiopterin-binding sites of neuronal nitric oxide synthase. Biochem J. 1997 Apr 1;323(Pt 1):131–139. doi: 10.1042/bj3230131. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Bredt D. S., Hwang P. M., Glatt C. E., Lowenstein C., Reed R. R., Snyder S. H. Cloned and expressed nitric oxide synthase structurally resembles cytochrome P-450 reductase. Nature. 1991 Jun 27;351(6329):714–718. doi: 10.1038/351714a0. [DOI] [PubMed] [Google Scholar]
  6. Chabin R. M., McCauley E., Calaycay J. R., Kelly T. M., MacNaul K. L., Wolfe G. C., Hutchinson N. I., Madhusudanaraju S., Schmidt J. A., Kozarich J. W. Active-site structure analysis of recombinant human inducible nitric oxide synthase using imidazole. Biochemistry. 1996 Jul 23;35(29):9567–9575. doi: 10.1021/bi960476o. [DOI] [PubMed] [Google Scholar]
  7. Chen P. F., Tsai A. L., Berka V., Wu K. K. Endothelial nitric-oxide synthase. Evidence for bidomain structure and successful reconstitution of catalytic activity from two separate domains generated by a baculovirus expression system. J Biol Chem. 1996 Jun 14;271(24):14631–14635. [PubMed] [Google Scholar]
  8. Cubberley R. R., Alderton W. K., Boyhan A., Charles I. G., Lowe P. N., Old R. W. Cysteine-200 of human inducible nitric oxide synthase is essential for dimerization of haem domains and for binding of haem, nitroarginine and tetrahydrobiopterin. Biochem J. 1997 Apr 1;323(Pt 1):141–146. doi: 10.1042/bj3230141. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Duch D. S., Bowers S. W., Nichol C. A. Elevation of brain histamine levels by diaminopyrimidine inhibitors of histamine N-methyl transferase. Biochem Pharmacol. 1978 May 15;27(10):1507–1509. doi: 10.1016/0006-2952(78)90109-0. [DOI] [PubMed] [Google Scholar]
  10. Furfine E. S., Harmon M. F., Paith J. E., Garvey E. P. Selective inhibition of constitutive nitric oxide synthase by L-NG-nitroarginine. Biochemistry. 1993 Aug 24;32(33):8512–8517. doi: 10.1021/bi00084a017. [DOI] [PubMed] [Google Scholar]
  11. Gachhui R., Ghosh D. K., Wu C., Parkinson J., Crane B. R., Stuehr D. J. Mutagenesis of acidic residues in the oxygenase domain of inducible nitric-oxide synthase identifies a glutamate involved in arginine binding. Biochemistry. 1997 Apr 29;36(17):5097–5103. doi: 10.1021/bi970331x. [DOI] [PubMed] [Google Scholar]
  12. Gachhui R., Presta A., Bentley D. F., Abu-Soud H. M., McArthur R., Brudvig G., Ghosh D. K., Stuehr D. J. Characterization of the reductase domain of rat neuronal nitric oxide synthase generated in the methylotrophic yeast Pichia pastoris. Calmodulin response is complete within the reductase domain itself. J Biol Chem. 1996 Aug 23;271(34):20594–20602. doi: 10.1074/jbc.271.34.20594. [DOI] [PubMed] [Google Scholar]
  13. Garvey E. P., Oplinger J. A., Furfine E. S., Kiff R. J., Laszlo F., Whittle B. J., Knowles R. G. 1400W is a slow, tight binding, and highly selective inhibitor of inducible nitric-oxide synthase in vitro and in vivo. J Biol Chem. 1997 Feb 21;272(8):4959–4963. doi: 10.1074/jbc.272.8.4959. [DOI] [PubMed] [Google Scholar]
  14. Garvey E. P., Oplinger J. A., Tanoury G. J., Sherman P. A., Fowler M., Marshall S., Harmon M. F., Paith J. E., Furfine E. S. Potent and selective inhibition of human nitric oxide synthases. Inhibition by non-amino acid isothioureas. J Biol Chem. 1994 Oct 28;269(43):26669–26676. [PubMed] [Google Scholar]
  15. Ghosh D. K., Stuehr D. J. Macrophage NO synthase: characterization of isolated oxygenase and reductase domains reveals a head-to-head subunit interaction. Biochemistry. 1995 Jan 24;34(3):801–807. doi: 10.1021/bi00003a013. [DOI] [PubMed] [Google Scholar]
  16. Gorman C., Skinner R. H., Skelly J. V., Neidle S., Lowe P. N. Equilibrium and kinetic measurements reveal rapidly reversible binding of Ras to Raf. J Biol Chem. 1996 Mar 22;271(12):6713–6719. doi: 10.1074/jbc.271.12.6713. [DOI] [PubMed] [Google Scholar]
  17. Gorren A. C., List B. M., Schrammel A., Pitters E., Hemmens B., Werner E. R., Schmidt K., Mayer B. Tetrahydrobiopterin-free neuronal nitric oxide synthase: evidence for two identical highly anticooperative pteridine binding sites. Biochemistry. 1996 Dec 24;35(51):16735–16745. doi: 10.1021/bi961931j. [DOI] [PubMed] [Google Scholar]
  18. Klatt P., Schmid M., Leopold E., Schmidt K., Werner E. R., Mayer B. The pteridine binding site of brain nitric oxide synthase. Tetrahydrobiopterin binding kinetics, specificity, and allosteric interaction with the substrate domain. J Biol Chem. 1994 May 13;269(19):13861–13866. [PubMed] [Google Scholar]
  19. Klatt P., Schmidt K., Brunner F., Mayer B. Inhibitors of brain nitric oxide synthase. Binding kinetics, metabolism, and enzyme inactivation. J Biol Chem. 1994 Jan 21;269(3):1674–1680. [PubMed] [Google Scholar]
  20. Knowles R. G., Moncada S. Nitric oxide synthases in mammals. Biochem J. 1994 Mar 1;298(Pt 2):249–258. doi: 10.1042/bj2980249. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. List B. M., Klatt P., Werner E. R., Schmidt K., Mayer B. Overexpression of neuronal nitric oxide synthase in insect cells reveals requirement of haem for tetrahydrobiopterin binding. Biochem J. 1996 Apr 1;315(Pt 1):57–63. doi: 10.1042/bj3150057. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Lowe P. N., Smith D., Stammers D. K., Riveros-Moreno V., Moncada S., Charles I., Boyhan A. Identification of the domains of neuronal nitric oxide synthase by limited proteolysis. Biochem J. 1996 Feb 15;314(Pt 1):55–62. doi: 10.1042/bj3140055. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Marletta M. A. Nitric oxide synthase: aspects concerning structure and catalysis. Cell. 1994 Sep 23;78(6):927–930. doi: 10.1016/0092-8674(94)90268-2. [DOI] [PubMed] [Google Scholar]
  24. Mayer B., Klatt P., Werner E. R., Schmidt K. Molecular mechanisms of inhibition of porcine brain nitric oxide synthase by the antinociceptive drug 7-nitro-indazole. Neuropharmacology. 1994 Nov;33(11):1253–1259. doi: 10.1016/0028-3908(94)90024-8. [DOI] [PubMed] [Google Scholar]
  25. Mayer B., Werner E. R. In search of a function for tetrahydrobiopterin in the biosynthesis of nitric oxide. Naunyn Schmiedebergs Arch Pharmacol. 1995 May;351(5):453–463. doi: 10.1007/BF00171035. [DOI] [PubMed] [Google Scholar]
  26. McMillan K., Masters B. S. Prokaryotic expression of the heme- and flavin-binding domains of rat neuronal nitric oxide synthase as distinct polypeptides: identification of the heme-binding proximal thiolate ligand as cysteine-415. Biochemistry. 1995 Mar 21;34(11):3686–3693. doi: 10.1021/bi00011a025. [DOI] [PubMed] [Google Scholar]
  27. Morrison J. F., Walsh C. T. The behavior and significance of slow-binding enzyme inhibitors. Adv Enzymol Relat Areas Mol Biol. 1988;61:201–301. doi: 10.1002/9780470123072.ch5. [DOI] [PubMed] [Google Scholar]
  28. Nishimura J. S., Martasek P., McMillan K., Salerno J., Liu Q., Gross S. S., Masters B. S. Modular structure of neuronal nitric oxide synthase: localization of the arginine binding site and modulation by pterin. Biochem Biophys Res Commun. 1995 May 16;210(2):288–294. doi: 10.1006/bbrc.1995.1659. [DOI] [PubMed] [Google Scholar]
  29. Riveros-Moreno V., Heffernan B., Torres B., Chubb A., Charles I., Moncada S. Purification to homogeneity and characterisation of rat brain recombinant nitric oxide synthase. Eur J Biochem. 1995 May 15;230(1):52–57. doi: 10.1111/j.1432-1033.1995.tb20533.x. [DOI] [PubMed] [Google Scholar]
  30. Sheta E. A., McMillan K., Masters B. S. Evidence for a bidomain structure of constitutive cerebellar nitric oxide synthase. J Biol Chem. 1994 May 27;269(21):15147–15153. [PubMed] [Google Scholar]
  31. Venema R. C., Ju H., Zou R., Ryan J. W., Venema V. J. Subunit interactions of endothelial nitric-oxide synthase. Comparisons to the neuronal and inducible nitric-oxide synthase isoforms. J Biol Chem. 1997 Jan 10;272(2):1276–1282. doi: 10.1074/jbc.272.2.1276. [DOI] [PubMed] [Google Scholar]
  32. Wolff D. J., Gribin B. J. The inhibition of the constitutive and inducible nitric oxide synthase isoforms by indazole agents. Arch Biochem Biophys. 1994 Jun;311(2):300–306. doi: 10.1006/abbi.1994.1241. [DOI] [PubMed] [Google Scholar]

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