Nitroarachidonic acid, a novel peroxidase inhibitor of prostaglandin endoperoxide H synthases 1 and 2.
Prostaglandin endoperoxide H synthase (PGHS) catalyzes the oxidation of arachidonate to prostaglandin H(2). We have previously synthesized and chemically characterized nitroarachidonic acid (AANO(2)), a novel anti-inflammatory signaling mediator. Herein, the interaction of AANO(2) with PGHS was analyzed. AANO(2) inhibited oxygenase activity of PGHS-1 but not PGHS-2. AANO(2) exhibited time- and concentration-dependent inhibition of peroxidase activity in both PGHS-1 and -2. The plot of k(obs) versus AANO(2) concentrations showed a hyperbolic function with k(inact) = 0.045 s(-1) and K(i)(*app) = 0.019 μM for PGHS-1 and k(inact) = 0.057 s(-1) and K(i)(*app) = 0.020 μM for PGHS-2. Kinetic analysis suggests that inactivation of PGHS by AANO(2) involves two sequential steps: an initial reversible binding event (described by K(i)) followed by a practically irreversible event (K(i)(*app)) leading to an inactivated enzyme. Inactivation was associated with irreversible disruption of heme binding to the protein. The inhibitory effects of AANO(2) were selective because other nitro-fatty acids tested, such as nitrooleic acid and nitrolinoleic acid, were unable to inhibit enzyme activity. In activated human platelets, AANO(2) significantly decreased PGHS-1-dependent thromboxane B(2) formation in parallel with a decrease in platelet aggregation, thus confirming the biological relevance of this novel inhibitory pathway.
Departamento de Bioquímica and Center for Free Radical and Biomedical Research, Facultad de Medicina, Montevideo, Uruguay.
Andrés
A
Trostchansky
Lucía
L
Bonilla
Christopher P
CP
Thomas
Valerie B
VB
O'Donnell
Lawrence J
LJ
Marnett
Rafael
R
Radi
Homero
H
Rubbo
D001095:Arachidonic Acids / Q000737:chemistry* / Q000494:pharmacology; D001792:Blood Platelets / Q000201:enzymology; D051545:Cyclooxygenase 1 / Q000737:chemistry* / Q000378:metabolism; D051546:Cyclooxygenase 2 / Q000737:chemistry* / Q000378:metabolism; D052246:Cyclooxygenase 2 Inhibitors / Q000737:chemistry* / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D007700:Kinetics; D009195:Peroxidase / Q000037:antagonists & inhibitors*; D010974:Platelet Aggregation / Q000187:drug effects; D011485:Protein Binding; D013929:Thromboxane B2 / Q000096:biosynthesis / Q000737:chemistry
D001095:Arachidonic Acids; D052246:Cyclooxygenase 2 Inhibitors; D013929:Thromboxane B2; D009195:Peroxidase; D051545:Cyclooxygenase 1; D051546:Cyclooxygenase 2; C496511:PTGS1 protein, human
Prostaglandins Leukot Essent Fatty Acids. 1996 Apr;54(4):229-38
8804119
J Biol Chem. 2008 Dec 26;283(52):36176-84
18974051
Science. 2000 Sep 15;289(5486):1933-7
10988074
FASEB J. 1999 Jul;13(10):1121-36
10385604
Adv Enzymol Relat Areas Mol Biol. 1988;61:201-301
3281418
J Biol Chem. 2002 Jan 4;277(1):478-85
11677234
Arch Biochem Biophys. 1985 Nov 15;243(1):125-34
4062299
Curr Vasc Pharmacol. 2004 Jan;2(1):81-93
15320836
Anal Biochem. 1987 Feb 15;161(1):1-15
3578775
Methods Enzymol. 1990;187:479-85
2122187
J Biol Chem. 1997 Apr 4;272(14):8885-94
9083007
Free Radic Biol Med. 2009 May 1;46(9):1250-9
19353781
J Biol Chem. 2009 Jan 16;284(3):1461-73
19015269
J Biol Chem. 1962 Oct;237:3245-9
14033211
Chem Rev. 2003 Jun;103(6):2239-304
12797830
Mol Pharmacol. 1984 Sep;26(2):328-35
6434940
Eur J Biochem. 1987 Jul 1;166(1):173-80
3036519
J Med Chem. 2007 Apr 5;50(7):1425-41
17341061
Prostaglandins. 1987 Aug;34(2):225-40
3118417
Biochemistry. 1996 Jul 16;35(28):9076-82
8703911
J Biol Chem. 2002 Feb 22;277(8):5832-40
11748216
Annu Rev Biochem. 2000;69:145-82
10966456
Prostaglandins Other Lipid Mediat. 2002 Aug;68-69:153-64
12432915
J Biol Chem. 2005 May 13;280(19):19289-97
15764811
J Biol Chem. 1994 Feb 25;269(8):5527-36
8119886
J Biol Chem. 1996 Dec 27;271(52):33157-60
8969167
Biochemistry. 2002 Dec 31;41(52):15451-8
12501173
J Biol Chem. 1999 Aug 13;274(33):22903-6
10438452
J Biol Chem. 1998 Apr 10;273(15):8903-9
9535872
J Biol Chem. 2001 Jun 8;276(23):19879-88
11279106
J Biol Chem. 2007 Jul 13;282(28):20151-63
17519227
J Biol Chem. 2000 Dec 8;275(49):38239-44
10993875
Biochemistry. 2007 Apr 17;46(15):4645-53
17373826
Biochem Biophys Res Commun. 2001 Dec 14;289(4):869-75
11735127
J Atheroscler Thromb. 2005;12(3):154-62
16020916
J Clin Invest. 1996 Jun 1;97(11):2562-8
8647949
Free Radic Biol Med. 2007 Apr 1;42(7):1029-38
17349930
Can J Comp Med. 1983 Apr;47(2):203-6
6883186
J Biol Chem. 2005 Dec 23;280(51):42464-75
16227625
Proc Natl Acad Sci U S A. 1996 Dec 24;93(26):15069-74
8986765
J Pharmacol Exp Ther. 2001 Nov;299(2):611-9
11602673
J Biol Chem. 2007 Oct 19;282(42):31085-93
17720974
J Biol Chem. 2006 Jul 21;281(29):20450-63
16682416
Am J Clin Nutr. 2006 Jun;83(6 Suppl):1505S-1519S
16841861