Nitric oxide synthases (NOS) are a family of enzymes that catalyze the production of nitric oxide (NO) from L-arginine. There are three main types of NOS - neuronal NOS, inducible NOS, and endothelial NOS - located in different tissues and performing different functions. NOS is a heme-containing dimerized protein that uses NADPH, FAD, FMN, tetrahydrobiopterin, and heme as cofactors. It converts L-arginine to citrulline and NO in a two-domain reaction involving the transfer of electrons from the reductase domain to the oxygenase domain. NOS and NO play important roles in many physiological processes and human diseases

1. NITRIC OXIDE SYNTHASESNITRIC OXIDE SYNTHASES
PRESENTED BY
SAJAL SEN
IIT KANPUR
M.Sc (2yr)

2. THE ENZYME & ITS ACTION IN BRIEFTHE ENZYME & ITS ACTION IN BRIEF
P. Mukherjee, M. Cinelli,
Chem. Soc. Rev., 2014, 43, 6814- 6838

3. WHAT ARE THEYWHAT ARE THEY??
• A FAMILY OF ENZYMES CATALYZING THE PRODUCTION OF
Nitric Oxide(NO) FROM L-ARGININE.
• A HEME PROTEIN SIMILAR TO CytochromeP-450.
• A HOMODIMER,Molecular Weight:135-150 KD/monomer.
REACTION:

4. WHY SO IMPORTANT!!!
Significance Of NO
 MOLECULE OF THE YEAR 1992 BY
JOURNAL "Science".
 NOBEL PRIZE(1998) FOR THE
DISCOVERY OF NO'S SIGNALLING ROLE
TO Robert F. Furchgott,Louis J.
Ignarro and Ferid Murad IN
PHYSIOLOGY & MEDICINE.
FUNCTION:
 NEUROTRANSMITTER BUT SLIGHTLY
DIFFERENT FROM OTHERS.
 REGULATION OF BLOOD
FLOW,VASODILATION.
 REGULATING
INFLAMMATION,WOUND-HEALING.
 IMPORTANT ROLE IN LONG TERM
MEMORY.
 PENILE ERECTION IN HUMAN
MALES(ROLE OF VIAGRA,CIALIS!!!)

5. CLASSIFICATION:
NAME LOCATION FUNCTION
Neuronal NOS (nNOS or
NOS1)
(155kD)
NERVOUS TISSUE
SKELETAL MUSCLE
CELL COMMUNICATION
Inducible NOS (iNOS or
NOS2)
(125kD)
IMMUNE SYSTEM
CARDIO-VASCULAR
SYSTEM
IMMUNE DEFENSE
AGAINST PATHOGENS
Endothelial NOS (eNOS
or NOS3 or cNOS)
(135kD)
ENDOTHELIUM VASODILATION
Bacterial NOS (bNOS)
VARIOUS
GRAM POSITIVE
BACTERIA
DEFENSE AGAINST
ANTIBIOTICS,
IMMUNE ATTACK
M. Marletta, The Journal Of Biological Chemistry, Vol 268, No:17,12231-2234

6. STRUCTURE
1.EACH MONOMER MAINLY CONSISTS OF TWO DOMAINS:
A)REDUCTASE
B)OXYGENASE
2.TWO DOMAINS ARE INTERLINKED BY CALMODULIN(CaM) WHICH ACTS AS A MOLECULAR
SWITCH.
3.TWO MONOMERS ARE AGAIN CONNECTED BY TETRAHEDRAL Zn(II) ION.
S. Daff, Nitric Oxide, Rev., 2010

7. REDUCTASE DOMAIN:
1.IT HAS THREE Co-FACTORS BOUND TO IT.
2.ITS MAIN ROLE IS TO DONATE ELECTRON TO THE OXYGENASE DOMAIN.
NADPH
(Nicotinamide adenine
dinucleotide phosphate)
FAD
(Flavin adenine dinucleotide)
FMN
(Flavin mononucleotide)

8. OXYGENASE DOMAIN:
IT CONTAINS THE ACTIVE SITE THAT BINDS WITH L-ARGININE AND CONVERTS IT
INTO CITRULINE & NO.
IT HAS TWO CO-FACTORS BOUND WITH IT:
H4B
(tetrahydrobiopterin)
HEME
LINKED WITH
S-CYSTEINE
G. Tennyson,S. J. Lippard, Chemistry & Biology,vol.18, issue 10, 1211-1220

9. HOW IT WORKS?HOW IT WORKS?
Schematic Diagram of
overall reaction pathway
Electrons are donated from
NADPH to the reductase domain
and then proceed via FAD and
FMN to the oxygenase domain.
There the electrons interact with
the heme iron and BH4 at the active
site in order to catalyze the reaction
of oxygen with L-arginine,
generating L-citrulline and NO as
products
MAIN REACTION
W. Alderton,R. Knowles,C. Cooper, Biochem.J., 2001, 357:593-615

10. MECHANISTIC SPECULATION:MECHANISTIC SPECULATION:
Chemical
mechanism of NO
synthesis assuming
an oxyferryl complex
as the active
monooxygenating
agent for both l-Arg
and NOHA.
S. Daff, Nitric Oxide,Rev., 2010

11. ACTIVITY MEASUREMENT:ACTIVITY MEASUREMENT:
Ultra Sensitive Assay for Nitric Oxide Synthase:Ultra Sensitive Assay for Nitric Oxide Synthase:
The traditional method for measuring nitric oxide synthase (NOS) activity is performed by radiochemicalThe traditional method for measuring nitric oxide synthase (NOS) activity is performed by radiochemical
assay that measures the conversion of L-[3H]arginine to L-[3H]citrulline.Ultrasensitive NOS Assay Kitassay that measures the conversion of L-[3H]arginine to L-[3H]citrulline.Ultrasensitive NOS Assay Kit
employs a NADPH recycling system to allow NOS to operate linearly for hours as nitric oxide-derivedemploys a NADPH recycling system to allow NOS to operate linearly for hours as nitric oxide-derived
nitrate and nitrite accumulate.NOS can be assayed spectrophotometrically by measuring thenitrate and nitrite accumulate.NOS can be assayed spectrophotometrically by measuring the
accumulation of its stable degradation products, nitrate and nitrite.Nitrate is converted to nitrite by theaccumulation of its stable degradation products, nitrate and nitrite.Nitrate is converted to nitrite by the
enzyme nitrate reductase (NaR), followed by quantization of nitrite using Griess Reagent.enzyme nitrate reductase (NaR), followed by quantization of nitrite using Griess Reagent.
OXFORD BIOMEDICAL RESEARCHOXFORD BIOMEDICAL RESEARCH

12. FUTURE SCOPES & CONCLUSION:FUTURE SCOPES & CONCLUSION:
NOS INHIBITOR DRUGS:NOS INHIBITOR DRUGS:
Both underproduction and overproduction of NO have been linked to various human
pathologies. Impaired NO bioavailability from eNOS and nNOS can lead to hypertension,
impotence, or atherosclerosis, whereas excess NO production by iNOS can cause inflammation,
rheumatoid arthritis, inflammatory bowel disease, immune-type diabetes, stroke, and
cancer.Therefore, it is not surprising that much effort has been made to find specific inhibitors of nitric
oxide synthases (NOS).
FEWFEW
OF THEOF THE
NOSNOS
INHIBITORSINHIBITORS
P. Mukherjee, M. Cinelli,
Chem. Soc. Rev., 2014, 43, 6814- 6838

13. 1.What is the three-dimensional structure of the reductase
domain and of the full-length enzyme, and thus how do the
haem and reductase domains interact?
2.Does the distribution between monomer and dimer vary in
cells or in vivo, and if so what is its significance in the
regulation of the three NOS isoforms?
3.What are the key roles played by the pterin cofactor, and is it
redox cycling between BH4 and BH3 during the NOS reaction?
4.What is the mechanism of NO formation from NHA (the
monooxygenase II reaction)?
SOME QUESTIONS YET TO BE ANSWERED:SOME QUESTIONS YET TO BE ANSWERED:
W. Alderton, R. Knowles, C. Cooper, Biochem.J., 2001,357: 593-615

14. THANK YOUTHANK YOU
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