Nitric oxide synthase joey klen
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Nitric Oxide Synthase Joey Klen. Introduction. Nitric oxide (NO) is an important signaling biomolecule and a cytotoxin. NO is produced by nitric oxide synthase (NOS) Neuronal ( nNOS ) Neuronal tissues; NO as a neurotransmitter Inducible ( iNOS ) Macrophages; NO as a cytotoxin

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Nitric Oxide Synthase Joey Klen

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Nitric oxide synthase joey klen

Nitric Oxide SynthaseJoey Klen


Introduction

Introduction

  • Nitric oxide (NO) is an important signaling biomolecule and a cytotoxin.

  • NO is produced by nitric oxide synthase (NOS)

    • Neuronal (nNOS)

      • Neuronal tissues; NO as a neurotransmitter

    • Inducible (iNOS)

      • Macrophages; NO as a cytotoxin

    • Endothelial (eNOS)

      • Endothelial cells; NO as a vasodilator


Reaction

Reaction

  • Catalizes a 2-step reaction from L-Arg to L-citrulline and NO

  • Two major domains:

    • Catalytic (oxygenase) domain:

      • Heme

      • L-Arginine

      • Tetrahydrobiopterin (H4B)

    • Reductase Domain

      • NADPH

      • FAD, FMN


Step 1 of mechanism

Step 1 of Mechanism

  • Conversion of L-Arg to Nw-hydroxy-L-arginine (NHA)

  • Similar to cytochrome P450

NADPH

H4B


Step 2 of mechanism

Step 2 of Mechanism

  • Conversion of NHA to

    L-citrulline and NO

  • Not like P450

NADPH

H4B


Heme binding

Heme Binding

  • Cys-415 binds to heme Fe

  • Tyr-706 binds to heme propionate oxygen

  • Trp-409 can p-stack with heme ring


L arginine nha binding

L-Arginine/NHA Binding

  • Glu-592 binds in three places

  • Pro-565 H-bonds to a H2O, which then binds to the guanidinium N of L-arg.

  • Hydroxylated N-H of NHA binds to O2 oxygen bound to heme Fe


H 4 b binding

H4B Binding

  • Carbonyl H-Bonds

    • Ser-334

    • Trp-678

    • Phe-691

  • Arg-596

  • Heme propionate group

H4B


Zinc tetrathiolate cluster

Zinc Tetrathiolate Cluster

  • NOS is only active as a homodimer; the monomeric form is inactive

  • The dimer is held together by a Zn2+ ion complexed to 2 pairs of Cys residues from each paired monomer

  • High [NO] can cause S-nitrosation of 2 Cys residues, which leads to release of Zn and formation of inactive NOS monomers


Sequence alignment

Sequence Alignment

Key:

Alpha Helices

Beta Sheets

Heme Binding Residues

L-Arg/NHA Binding Residues

H4B Binding Residues

Zn Tetrathiolate Cys Residues


Alignment continued

Alignment Continued


Kinetic data

Kinetic Data

A. Rate of NO production of WT rat nNOS monitored by Hb assay at room temperature.

B. Lineweaver-Burk plot for dtermination of Km and Vmax.


References

References

  • (Structure Paper) Doukov, T., Li, H., Soltis, M., and Poulos, T. L. (2009) Single crystal structure and absorption spectral characterizations of nitric oxide synthase complexed with Nw-hydroxy-L-arginine and diatomic ligands. Biochemistry 48, 10246-10254.

  • Kerwin, J. R. Jr., Lancaster, J. R. Jr., and Feldman, P. L. (1995) Nitric oxide: a new paradigm for second messengers. J. Med. Chem. 38, 4343-4362.

  • Mitchell, D. A., Erwin, P. A., Michel, T., and Marletta, M. A. (2005) S-Nitrosation and regulation of inducible nitric oxide synthase. Biochemistry 44, 4636-4647.

  • Raman, C. S., Li, H., Martasek, P., Kral, V., Masters, B. S. S., and Poulos, T. L. (1998) Crystal structure of constitutive endothelial nitric oxide synthase: a paradigm for pterin function involving a novel metal center. Cell 95, 939-950.

  • Li, D., Kabir, M., Stuehr, D. J., Rousseau, D. L, and Yeh, S. R. (2007) Substrate- and isoform-specific dioxygen complexes of nitric oxide synthase. J. Am. Chem. Soc. 129, 6943-6951.

  • Fang, J., Ji, H., Lawton, G. R., Xue, F., Roman, L. J., and Silverman, R. B. (2009) L337H Mutant of rat neuronal nitric oxide synthase resembles human neuronal nitric oxide synthase toward inhibitors. J. Med. Chem. 52, 4533-4537


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