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NITRIC OXIDE SYNTHASESNITRIC OXIDE SYNTHASES
PRESENTED BYSAJAL SEN
IIT KANPURM.Sc (2yr)
THE ENZYME & ITS ACTION IN BRIEFTHE ENZYME & ITS ACTION IN BRIEF
P. Mukherjee, M. Cinelli,Chem. Soc. Rev., 2014, 43, 6814- 6838
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:
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!!!)
CLASSIFICATION:
NAME LOCATION FUNCTION
Neuronal NOS (nNOS or NOS1)
(155kD)
NERVOUS TISSUESKELETAL MUSCLE
CELL COMMUNICATION
Inducible NOS (iNOS or NOS2)
(125kD)
IMMUNE SYSTEMCARDIO-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
STRUCTURE
1.EACH MONOMER MAINLY CONSISTS OF TWO DOMAINS:A)REDUCTASEB)OXYGENASE2.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
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)
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)
HEMELINKED WITH S-CYSTEINE
G. Tennyson,S. J. Lippard, Chemistry & Biology,vol.18, issue 10, 1211-1220
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
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
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 radiochemical The 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 Kit assay 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-derived employs 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 the nitrate 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 the accumulation 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
FUTURE SCOPES & CONCLUSION:FUTURE SCOPES & CONCLUSION:
NOS INHIBITOR DRUGS:NOS INHIBITOR DRUGS:Both underproduction and overproduction of NO have been linked to various humanpathologies. 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, andcancer.Therefore, it is not surprising that much effort has been made to find specific inhibitors of nitric oxide synthases (NOS).
FEW FEW OF THE OF THE
NOSNOS
INHIBITORS INHIBITORS
P. Mukherjee, M. Cinelli,Chem. Soc. Rev., 2014, 43, 6814- 6838
1.What is the three-dimensional structure of the reductasedomain and of the full-length enzyme, and thus how do thehaem and reductase domains interact?
2.Does the distribution between monomer and dimer vary incells or in vivo, and if so what is its significance in theregulation of the three NOS isoforms?
3.What are the key roles played by the pterin cofactor, and is itredox cycling between BH4 and BH3 during the NOS reaction?
4.What is the mechanism of NO formation from NHA (themonooxygenase 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
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