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• W2102657944 abstract "Nitric-oxide synthase (NOS) catalyzes the oxidation of l-arginine to nitric oxide andl-citrulline. Because overproduction of nitric oxide causes tissue damage in neurological, inflammatory, and autoimmune disorders, design of NOS inhibitors has received much attention. Most inhibitors described to date include a guanidine-like structural motif and interact with the guanidinium region of thel-arginine-binding site. We report here studies withl-arginine analogs having one or both terminal guanidinium nitrogens replaced by functionalities that preserve some, but not all, of the molecular interactions possible for the –NH2, =NH, or =NH2+ groups of l-arginine. Replacement groups include –NH-alkyl, –alkyl, =O, and =S. Binding ofl-canavanine, an analog unable to form hydrogen bonds involving a N 5-proton, was also examined. From our results and previous work, we infer the orientation of these compounds in the l-arginine-binding site and use IC50 or Ki values and optical difference spectra to quantitate their affinity relative tol-arginine. We find that the non-reactive guanidinium nitrogen of l-arginine binds in a pocket that is relatively intolerant of changes in the size or hydrogen bonding properties of the group bound. The individual H-bonds involved are, however, weaker than expected (<2 versus 3–6 kcal). These findings elucidate substrate binding forces in the NOS active site and identify an important constraint on NOS inhibitor design. Nitric-oxide synthase (NOS) catalyzes the oxidation of l-arginine to nitric oxide andl-citrulline. Because overproduction of nitric oxide causes tissue damage in neurological, inflammatory, and autoimmune disorders, design of NOS inhibitors has received much attention. Most inhibitors described to date include a guanidine-like structural motif and interact with the guanidinium region of thel-arginine-binding site. We report here studies withl-arginine analogs having one or both terminal guanidinium nitrogens replaced by functionalities that preserve some, but not all, of the molecular interactions possible for the –NH2, =NH, or =NH2+ groups of l-arginine. Replacement groups include –NH-alkyl, –alkyl, =O, and =S. Binding ofl-canavanine, an analog unable to form hydrogen bonds involving a N 5-proton, was also examined. From our results and previous work, we infer the orientation of these compounds in the l-arginine-binding site and use IC50 or Ki values and optical difference spectra to quantitate their affinity relative tol-arginine. We find that the non-reactive guanidinium nitrogen of l-arginine binds in a pocket that is relatively intolerant of changes in the size or hydrogen bonding properties of the group bound. The individual H-bonds involved are, however, weaker than expected (<2 versus 3–6 kcal). These findings elucidate substrate binding forces in the NOS active site and identify an important constraint on NOS inhibitor design. nitric-oxide synthase neuronal NOS endothelial NOS inducible NOS nitric oxide N ω-hydroxy-l-arginine N ω-methyl-l-arginine N 5-(1-iminoethyl)-l-ornithine N 5-(1-iminobutyl)-l-ornithine N 5-(1-iminohexyl)-l-ornithine N 5-thioacetyl-l-ornithine N 5-thiobutyryl-l-ornithine N5-thiohexanoyl-l-ornithine dissociation constant determined from spectral studies electron impact fast atom bombardment-mass spectroscopy Nitric-oxide synthase (NOS)1 catalyzes the two-step oxidation of l-arginine to l-citrulline and nitric oxide (NO). Oxygen and NADPH are co-substrates, and N ω-hydroxy-l-arginine (NOH-Arg) is a tightly bound intermediate (1Griffith O.W. Stuehr D.J. Annu. Rev. Physiol. 1995; 57: 707-736Crossref PubMed Google Scholar, 2Stuehr D.J. Annu. Rev. Pharmacol. Toxicol. 1997; 37: 339-359Crossref PubMed Scopus (447) Google Scholar). The enzyme is active as a homodimer, and each monomer is comprised of a heme- and tetrahydrobiopterin-containing oxygenase domain that binds and oxidizesl-arginine and a FAD- and FMN-containing reductase domain that delivers electrons from NADPH to heme. Once reduced, the heme cofactor binds and activates O2, which in turn reacts with a terminal guanidinium nitrogen of the substrate l-arginine that is bound ∼4 Å from the heme iron (3Tierney D.L. Martasek P. Doan P.E. Masters B.S.S. Hoffman B.M. J. Am. Chem. Soc. 1998; 120: 2983-2984Crossref Google Scholar). That reactive, “proximal” nitrogen is first hydroxylated, forming NOH-Arg, and then oxidized further to NO. The other, previously equivalent guanidinium nitrogen is bound farther away from the heme cofactor and does not react; that “distal” nitrogen becomes the terminal –NH2 group of the product l-citrulline.There are three major isoforms of NOS in mammals (1Griffith O.W. Stuehr D.J. Annu. Rev. Physiol. 1995; 57: 707-736Crossref PubMed Google Scholar, 2Stuehr D.J. Annu. Rev. Pharmacol. Toxicol. 1997; 37: 339-359Crossref PubMed Scopus (447) Google Scholar, 4Sessa W.C. J. Vasc. Res. 1994; 31: 131-143Crossref PubMed Scopus (401) Google Scholar). Two constitutive, Ca2+/calmodulin-regulated isoforms were initially identified in neurons (nNOS) and vascular endothelial cells (eNOS). An inducible, transcriptionally regulated isoform (iNOS) was initially identified in macrophages but can be expressed in response to inflammatory cytokines and endotoxin in many cell types. Neuronal NOS has a role in neurotransmission and/or neuromodulation (5Garthwaite J. Boulton C.L. Annu. Rev. Physiol. 1995; 57: 683-706Crossref PubMed Scopus (1533) Google Scholar, 6Zhang J. Snyder S.H. Annu. Rev. Phamacol. Toxicol. 1995; 35: 213-233Crossref PubMed Scopus (354) Google Scholar), whereas eNOS produces NO that has an important role in controlling vasorelaxation and blood pressure (7Aisaka K. Gross S.S. Griffith O.W. Levi R. Biochem. Biophys. Res. Commun. 1989; 160: 881-886Crossref PubMed Scopus (378) Google Scholar, 8Umans J.G. Levi R. Annu. Rev. Physiol. 1995; 57: 771-790Crossref PubMed Scopus (249) Google Scholar). Nitric oxide derived from iNOS plays both regulatory and cytotoxic roles in the immune response (9Clancy R.M. Amin A.R. Abramson S.B. Arthritis Rheum. 1998; 41: 1141-1151Crossref PubMed Scopus (393) Google Scholar, 10Nathan C.F. Hibbs Jr., J.B. Curr. Opin. Immunol. 1991; 3: 65-70Crossref PubMed Scopus (1321) Google Scholar). In addition to these physiological roles, NOS is known to contribute to several pathological processes, typically when nNOS is overstimulated or iNOS is induced inappropriately or in excess. For example, nNOS is implicated in stroke (11Huang Z. Huang P.L. Panahian N. Dalkara T. Fishman M.C. Mosowitz M.A. Science. 1994; 265: 1883-1885Crossref PubMed Scopus (1449) Google Scholar) and migraine headache (12Lassen L.H. Ashina M. Christiansen I. Ulrich V. Olesen J. Lancet. 1997; 349: 401-402Abstract Full Text Full Text PDF PubMed Scopus (218) Google Scholar), and iNOS is implicated in septic shock (13Kilbourn R.G. Griffith O.W. J. Natl. Cancer Inst. 1992; 84: 827-831Crossref PubMed Scopus (260) Google Scholar, 14Kilbourn R.G. Junran A. Gross S.S. Griffith O.W. Levi R. Adams J. Lodato R.F. Biochem. Biophys. Res. Commun. 1990; 172: 1132-1138Crossref PubMed Scopus (556) Google Scholar), inflammatory bowel disease (15Guslandi M. Eur. J. Clin. Invest. 1998; 28: 904-907Crossref PubMed Scopus (94) Google Scholar), uveitis (16Goureau O. Belot J. Thillaye B. Courtois Y. de Kozak Y. J. Immunol. 1995; 154: 6518-6523PubMed Google Scholar), and arthritis (17McCartney-Francis N. Allen J.B. Mizel D.E. Albina J.E. Xie Q-W. Nathan C.F. Wahl S.M. J. Exp. Med. 1993; 178: 749-754Crossref PubMed Scopus (594) Google Scholar, 18Stefanovic-Racic M. Stadler J. Evans C.H. Arthritis Rheum. 1993; 36: 1036-1044Crossref PubMed Scopus (279) Google Scholar). The possibility of treating these and other conditions by inhibiting NOS has elicited intense efforts to identify or design NOS inhibitors, preferably isoform-selective NOS inhibitors. To date well over 100 inhibitors have been reported (19Griffith O.W. Gross S.S. Feelisch M. Stamler J.S. Methods in Nitric Oxide Research. John Wiley & Sons Ltd., New York1996: 187-208Google Scholar, 20Babu B.R. Griffith O.W. Curr. Opin. Chem. Biol. 1998; 2: 491-500Crossref PubMed Scopus (130) Google Scholar, 21Southan G.J. Szabó C. Biochem. Pharmacol. 1996; 51: 383-394Crossref PubMed Scopus (539) Google Scholar, 22Fukuto J.M. Chaudhuri G. Annu. Rev. Pharmacol. Toxicol. 1995; 35: 165-194Crossref PubMed Scopus (137) Google Scholar). Almost all of these compounds contain a guanidine-like structural motif, and their initial binding is competitive with l-arginine, observations that suggest they are interacting with the guanidinium region of thel-arginine-binding site.In the present studies we have designed and synthesized several novell-arginine analogs (Fig. 1) in order to “map” the guanidinium region of the substrate-binding site. We have confirmed and extended previous work showing that the binding site for the reactive (i.e. oxidizable) guanidinium nitrogen can accommodate a variety of alternative groups including those much larger than –NH2 and =NOH. In contrast, the binding site for the non-reactive, distal guanidinium nitrogen does not accommodate larger groups (1Griffith O.W. Stuehr D.J. Annu. Rev. Physiol. 1995; 57: 707-736Crossref PubMed Google Scholar, 23Hibbs Jr., J.B. Vavrin Z. Taintor R.R. J. Immunol. 1987; 138: 550-565PubMed Google Scholar, 24Griffith O.W. Kilbourn R.G. Adv. Enzymol. Regul. 1997; 37: 171-194Crossref PubMed Scopus (12) Google Scholar). We have exploited this difference in size specificity to predictably direct the binding orientation of novell-arginine analogs. We find that analogs bind poorly if the binding site for the non-reactive guanidinium nitrogen ofl-arginine must be occupied by =O, =S, –CH3 or larger groups. These results extend insights gained from recently reported mutagenesis and x-ray crystallographic studies of NOS and have implications for the design of NOS inhibitors.EXPERIMENTAL PROCEDURESMaterialsReagents for organic synthesis were obtained from Aldrich and biochemicals were obtained from Sigma, respectively. (6 R)-5,6,7,8-Tetrahydrobiopterin was purchased from Alexis (La Jolla, CA). l-[U-14C]Arginine was from NEN Life Science Products Inc. (Boston, MA).N 5-(1-Iminoethyl)-l-ornithine (l-NIO) (25Scannell J.P. Ax H.A. Pruess D.L. Williams T. Demny T.C. Stempel A. J. Antibiot. 1972; 25: 179-184Crossref PubMed Scopus (29) Google Scholar) and N 5-acetyl-l-ornithine (26Neuberger A. Sanger F. Biochem. J. 1943; 37: 515-518Crossref PubMed Google Scholar) were prepared by the general methods indicated.Rat nNOS for most studies was isolated from stably transfected kidney 293 cells as described (27McMillan K. Bredt D.S. Hirsch D.J. Snyder S.H. Clark J.E. Masters B.S.S. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 11141-11145Crossref PubMed Scopus (354) Google Scholar). Bovine eNOS (28Martásek P. Liu Q. Liu J. Roman L.J. Gross S.S. Sessa W.C. Masters B.S.S. Biochem. Biophys. Res. Commun. 1996; 219: 359-365Crossref PubMed Scopus (142) Google Scholar, 29Vásquez-Vivar J. Kalyanaraman B. Martásek P. Hogg N. Masters B.S.S. Karoui H. Tordo P. Pritchard Jr., K.A. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 9220-9225Crossref PubMed Scopus (1215) Google Scholar) and mouse iNOS (30Xia Y. Roman L.J. Masters B.S.S. Zweier J.L. J. Biol. Chem. 1998; 273: 22635-22639Abstract Full Text Full Text PDF PubMed Scopus (344) Google Scholar), both expressed in Escherichia coli, were generous gifts from Dr. Kirkwood Pritchard (Department of Pathology, Medical College of Wisconsin, Milwaukee, WI) and Drs. Linda J. Roman and Bettie S. S. Masters (Department of Biochemistry, University of Texas Health Science Center, San Antonio, TX), respectively. The latter investigators also provided rat nNOS isolated from transfected E. coli (31Roman L.J. Sheta E.A. Martásek P. Gross S.S. Liu Q. Masters B.S.S. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 8428-8432Crossref PubMed Scopus (244) Google Scholar).Methods for Synthesis of Arginine Analogs1H and 13C NMR spectra were obtained using a Bruker AC 300 MHz spectrometer. High-resolution, electron impact (EI), chemical ionization, and FAB mass spectral analyses were generously carried out by Dr. Frank Laib at the Department of Chemistry, University of Wisconsin, Milwaukee, WI.Syntheses of N5-Thioacyl-l-ornithinesN 5-Thioacetyl-l-ornithine (TAO) was synthesized by reaction of an appropriately protectedl-ornithine with ethyl dithioacetate. Thus, N α-(tert-butyloxycarbonyl)-l-ornithine tert-butyl ester (2.9 g, 10 mmol) was dissolved in 50 ml of chloroform and added to a solution of 2.5 g of CaCO3and 1.20 ml of ethyl dithioacetate dissolved in 50 ml of water. The mixture was stirred vigorously for 20 h and filtered. The chloroform layer was separated, and the aqueous layer was extracted with chloroform (2 × 50 ml). The combined chloroform extracts were dried with anhydrous Na2SO4 and evaporated under reduced pressure. The residue was dissolved in a small amount of ethyl acetate (5 ml) and chromatographed over a silica gel column (3 × 50 cm) eluted with ethyl acetate and petroleum ether (1:4). Fractions (5 ml) were collected and the product was identified by TLC on silica plates developed in the same solvent (RF = 0.8). Product-containing fractions were pooled and solvent was evaporated under reduced pressure to give N α-(tert-butyloxycarbonyl)-N 5-(thioacetyl)-l-ornithine tert-butyl ester as an oily liquid in 25% yield. That intermediate was dissolved in ∼10 ml of dioxane and added to ∼20 ml of ice-cold dioxane containing 6 n HCl. After stirring on ice for 4 h and at room temperature overnight, evaporation of the solvent under reduced pressure yielded 0.4 g of TAO (m.p. 75–78 °C): 1H NMR (D2O): δ 1.75–2.1 (m, 4H), 2.5 (s, 3H), 3.67 (t, 2H) and 4.09 (t, 1H); 13C NMR (D2O): δ 25.36, 29.89, 35.01, 47.88, 55.51, 174.98 and 203.75; MS (70 eV, EI): m/e 190 (M+); high resolution MS, m/e, C7H14N2O2S, Calculated: 190.0776, Found: 190.0763.N 5-Thiobutyryl-l-ornithine (TBO) was synthesized by reaction of Lawesson's reagent with N 5-butyryl-l-ornithine, which was obtained by reacting butyryl chloride with protectedl-ornithine. Thus, butyryl chloride (2.13 g, 20 mmol) was added to a solution of N α-(tert-butyloxycarbonyl)-l-ornithine tert-butyl ester (5.8 g, 20 mmol) in 100 ml of methylene chloride containing 2.0 ml of triethylamine. The reaction mixture was stirred at room temperature for 20 h, and the solvent was then evaporated under reduced pressure. The residue was chromatographed over silica gel as described for TAO.N α-(tert-Butyloxycarbonyl)-N 5-(butyryl)-l-ornithine tert-butyl ester (3.2 g, 45%) was obtained as an oily liquid. That product (8.0 mmol) was dissolved in benzene (50 ml), Lawesson's reagent (1.82 g, 4.5 mmol) was added, and the mixture was refluxed for 3 h. After cooling, the mixture was filtered, and the filtrate was washed with water (3 × 50 ml). The benzene layer was dried over anhydrous Na2SO4 and evaporated to give an oily liquid that was chromatographed over silica gel as described previously. Product-containing fractions were determined by TLC (RF = 0.8) and evaporated to give N α-(tert-butyloxycarbonyl)-N 5-(thiobutyryl)-l-ornithine tert-butyl ester (2.5 g, 85%) as an oily liquid. Deprotection of that intermediate in dioxane-dry HCl as described above gave 1.1 g of TBO (80% yield) as colorless crystalline solid (m.p. 225–227 °C): 1H NMR (D2O): δ 0.87 (t, 3H), 1.72 (q, 2H), 1.76–2.05 (m, 4H), 2.62 (t, 2H), 3.66 (t, 2H) and 4.07 (t, 1H); 13C NMR (D2O): δ 14.99, 25.04, 25.49, 29.95, 47.56, 50.18, 55.48, 174.86 and 208.36; MS (70 eV, EI): m/e 218 (M+); high resolution MS, m/e C9H18N2O2S, Calculated: 218.1089, Found: 218.1089.N 5-Thiohexanoyl-l-ornithine (THO) was synthesized as described for TBO except hexanoyl chloride was used in place of butyryl chloride (m.p. 200–205 °C): 1H NMR (D2O) δ 0.87 (t, 3H), 1.30 (m, 4H), 1.71 (m, 2H), 1.71–2.1 (m, 4H), 2.67 (t, 2H), 3.68 (t, 2H) and 4.09 (t, 1H);13C NMR (D2O): δ 15.85, 24.35, 25.43, 29.97, 31.25, 32.78, 38.47, 47.69, 55.48, 175.10 and 208.30; MS (70 eV, EI):m/e 246 (M+); high resolution MS, m/e C11H22N2O2S, Calculated: 246.1402, Found: 246.1402.Synthesis of N5-(1-Iminoalkyl)-l-ornithinesThe following compounds, which are homologs of l-NIO, were synthesized by the general procedure reported previously for N 5-(1-iminopropyl)-l-ornithine (methyl-l-NIO) (32Babu B.R. Griffith O.W. J. Biol. Chem. 1998; 273: 8882-8889Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar).N 5-(1-Iminobutyl)-l-ornithine (ethyl-l-NIO): m.p. 144–147 °C (dec); 1H NMR (D2O): δ 0.93 (t, 3H), 1.6–2.0 (m, 6H), 2.43 (t, 2H), 3.3 (t, 2H) and 3.75 (t, 1H); 13C NMR (D2O): δ 14.94, 22.64, 25.37, 30.33, 37.07, 44.01, 56.90, 170.72, and 176.99; FAB-MS: m/e 202 (M + H).N 5-(1-Iminohexyl)-l-ornithine (butyl-l-NIO): m.p. 130–134 °C (dec); 1H NMR (D2O): δ 0.90 (t, 3H), 1.30 (m, 4H), 1.5–2.05 (m, 6H), 2.5 (t, 2H), 3.33 (t, 2H) and 3.8 (t, 1H); 13C NMR (D2O): δ 15.76, 24.20, 25.39, 28.85, 32.71, 37.30, 44.03, 56.89, 61.12, 171.01 and 176.92; FAB-MS: m/e 230 (M + H).Synthesis of N5-Acyl-l-ornithinesThese derivatives were synthesized by dioxane-HCl deprotection of the corresponding N α-(tert-butyloxycarbonyl)-N 5-acyl-l-ornithine tert-butyl esters which were in turn prepared from N α-(tert-butyloxycarbonyl)-l-ornithine tert-butyl ester and the appropriate acyl chloride as described above for N α-(tert-butyloxycarbonyl)-N 5-butyryl-l-ornithine tert-butyl ester.N 5-Butyryl-l-ornithine: m.p. 65–69 °C; 1H NMR (D2O): δ 0.90 (t, 3H), 1.5–2.09 (m, 6H), 2.25 (t, 2H), 3.25 (t, 2H), and 4.07 (t, 1H);13C NMR (D2O): δ 15.34, 21.68, 26.82, 29.59, 41.02, 55.48, 69.23, 174.53, and 180.10; MS (chemical ionization):m/e 203 (M + H).N 5-Hexanoyl-l-ornithine: m.p. 78–80 °C; 1H NMR (D2O): δ 0.85 (t, 3H), 1.26 (m, 4H), 1.5–2.05 (m, 6H), 2.2 (t, 2H), 3.23 (t, 2H), and 4.09 (t, 1H); 13C NMR (D2O): δ 15.85, 24.32, 27.76, 28.78, 33.10, 38.44, 41.38, 55.19, 69.22, 174.53, and 180.12; MS (chemical ionization): m/e 231 (M + H).Methods for Enzymatic StudiesNitric-oxide Synthase AssaysNitric-oxide synthase activity was routinely determined based on the oxidation of oxyhemoglobin to methemoglobin by NO (33Feelisch M. Noack E.A. Eur. J. Pharmacol. 1987; 139: 19-30Crossref PubMed Scopus (838) Google Scholar). Sample cuvettes at 25 °C contained in a final volume of 0.5 ml, 50 mm Hepes buffer, pH 7.4, 0.1 mm EDTA, 50 μm tetrahydrobiopterin, 10 μg/ml calmodulin, 0.2 mm CaCl2, 0.1 mm glutathione, 1.0 μm FAD, 1.0 μm FMN, 1 mg/ml bovine serum albumin, 0.5 mmNADPH, 20 μml-arginine, and 5 μm bovine oxyhemoglobin (prepared by reduction with sodium dithionite followed by gel filtration). Formation of methemoglobin was monitored at 401 nm (ε = 0.038 μm−1) (33Feelisch M. Noack E.A. Eur. J. Pharmacol. 1987; 139: 19-30Crossref PubMed Scopus (838) Google Scholar); the reference cuvette contained a similar mixture without enzyme.IC50 and Ki DeterminationsFor determination of most inhibition constants NOS activity was measured by following the conversion of l-[14C]arginine to l-[14C]citrulline (32Babu B.R. Griffith O.W. J. Biol. Chem. 1998; 273: 8882-8889Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar). Reaction mixtures contained in a final volume of 50 μl, 50 mmNa+ Hepes buffer, pH 7.4, 100 μm EDTA, 0.2 mm CaCl2, 10 μg/ml calmodulin, 100 μm dithiothreitol, 50 μmtetrahydrobiopterin, 1.0 μm FAD, 1.0 μmFMN, 100 μg/ml bovine serum albumin, 500 μm NADPH, and various concentrations of l-[14C]arginine and inhibitor. Reaction was initiated by the addition of NOS, and mixtures were maintained at 25 °C for 4 min. Reaction mixtures were then quenched by addition of 200 μl of stop buffer (100 mmNa+ Hepes buffer, pH 5.5, and 5 mm EGTA). After heating in a boiling water bath for 1 min, the samples were chilled and centrifuged. A portion (225 μl) of the supernatant was applied to small Dowex 50 columns (Na+ form, 1 ml of resin), and the product l-[14C]citrulline was eluted with 2 ml of water and quantitated by liquid scintillation counting. Where inhibition was determined to be competitive withl-arginine, Ki values were estimated from measured IC50 values using Ki = IC50(KmArg/(KmArg+ [Arg])). For purpose of calculation, KmArg values for nNOS, eNOS, and iNOS were estimated as 1.8, 3.6, and 12.5 μm, respectively, based on the present and earlier (32Babu B.R. Griffith O.W. J. Biol. Chem. 1998; 273: 8882-8889Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar) work.Optical Difference SpectroscopyInteraction of inhibitors with nNOS was determined spectrally using a Shimadzu or Perkin-Elmer dual beam UV/visible spectrophotometer (32Babu B.R. Griffith O.W. J. Biol. Chem. 1998; 273: 8882-8889Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar). Typically 1.5–2.0 μm nNOS in 0.5 ml of 50 mm Tris-HCl buffer, pH 7.5, 10% glycerol, and 0.1 mm EDTA was placed in the sample and reference cuvettes at 15 °C, and the baseline was adjusted to zero. Inhibitor was then added to the sample cuvette, an equal volume of the same buffer was added to reference cuvette, and the difference spectrum was obtained. Spectra were normalized using the isosbestic point at 410 nm.DISCUSSIONHigh resolution x-ray crystallographic structures for the oxygenase domains of all 3 mammalian NOS isoforms have now been reported in publications (49Crane B.R. Arvai A.S. Ghosh D.K. Wu C. Getzoff E.D. Stuehr D.J. Tainer J.A. Science. 1998; 279: 2121-2126Crossref PubMed Scopus (621) Google Scholar, 50Raman C.S. Li H. Martásek P. Kral V. Masters B.S.S. Poulos T.L. Cell. 1998; 95: 939-950Abstract Full Text Full Text PDF PubMed Scopus (571) Google Scholar, 51Fischmann T.O. Hruza A. Niu X.D. Fossetta J.D. Lunn C.A. Dolphin E. Prongay A.J. Reichert P. Lundell D.J. Narula S.K. Weber P.C. Nature Struct. Biol. 1999; 6: 233-242Crossref PubMed Scopus (400) Google Scholar) or at meetings. As expected, the active sites show a high degree of homology, but there are subtle differences (49Crane B.R. Arvai A.S. Ghosh D.K. Wu C. Getzoff E.D. Stuehr D.J. Tainer J.A. Science. 1998; 279: 2121-2126Crossref PubMed Scopus (621) Google Scholar, 50Raman C.S. Li H. Martásek P. Kral V. Masters B.S.S. Poulos T.L. Cell. 1998; 95: 939-950Abstract Full Text Full Text PDF PubMed Scopus (571) Google Scholar, 51Fischmann T.O. Hruza A. Niu X.D. Fossetta J.D. Lunn C.A. Dolphin E. Prongay A.J. Reichert P. Lundell D.J. Narula S.K. Weber P.C. Nature Struct. Biol. 1999; 6: 233-242Crossref PubMed Scopus (400) Google Scholar, 52Fan B. Wang J. Stuehr D.J. Rousseau D.L. Biochemistry. 1997; 36: 12660-12665Crossref PubMed Scopus (66) Google Scholar). Consistent with conclusions from substrate specificity (20Babu B.R. Griffith O.W. Curr. Opin. Chem. Biol. 1998; 2: 491-500Crossref PubMed Scopus (130) Google Scholar, 37Fasehun O. Gross S.S. Pipili E. Jaffe E. Griffith O.W. Levi R. FASEB J. 1990; 4 (abstr.): A309Google Scholar) and ENDOR (3Tierney D.L. Martasek P. Doan P.E. Masters B.S.S. Hoffman B.M. J. Am. Chem. Soc. 1998; 120: 2983-2984Crossref Google Scholar) studies, the distal, non-reactive guanidinium nitrogen of l-arginine is found to bind >4 Å from heme iron in a sterically constrained pocket 3In eNOS the distance between heme iron and the distal guanidinium and the N 5 nitrogens are 4.7 and 4.9 Å, respectively (Ref. 50Raman C.S. Li H. Martásek P. Kral V. Masters B.S.S. Poulos T.L. Cell. 1998; 95: 939-950Abstract Full Text Full Text PDF PubMed Scopus (571) Google Scholar, and C. S. Raman, personal communication).; a conserved enzymatic glutamate residue, identified earlier in mutagenesis studies (53Gachhui R. Ghosh D.K. Wu C. Parkinson J. Crane B.R. Stuehr D.J. Biochemistry. 1997; 36: 5097-5103Crossref PubMed Scopus (72) Google Scholar, 54Chen P.-F. Tsai A-L. Berka V. Wu K.K. J. Biol. Chem. 1997; 272: 6114-6118Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar), forms H-bonds to protons on the distal guanidinium nitrogen and the N 5 nitrogen of l-arginine. Unanticipated from earlier work, binding of the distal guanidinium nitrogen of l-arginine is also stabilized by an H-bond to the backbone carbonyl of a conserved tryptophan residue (Fig.6) (49Crane B.R. Arvai A.S. Ghosh D.K. Wu C. Getzoff E.D. Stuehr D.J. Tainer J.A. Science. 1998; 279: 2121-2126Crossref PubMed Scopus (621) Google Scholar, 50Raman C.S. Li H. Martásek P. Kral V. Masters B.S.S. Poulos T.L. Cell. 1998; 95: 939-950Abstract Full Text Full Text PDF PubMed Scopus (571) Google Scholar, 51Fischmann T.O. Hruza A. Niu X.D. Fossetta J.D. Lunn C.A. Dolphin E. Prongay A.J. Reichert P. Lundell D.J. Narula S.K. Weber P.C. Nature Struct. Biol. 1999; 6: 233-242Crossref PubMed Scopus (400) Google Scholar). The present studies further elucidate the importance of these interactions to substrate and inhibitor binding.Figure 6Proposed binding interactions ofl-arginine and its analogs with the NOS active site. The top line shows the normal NOS reaction (49Crane B.R. Arvai A.S. Ghosh D.K. Wu C. Getzoff E.D. Stuehr D.J. Tainer J.A. Science. 1998; 279: 2121-2126Crossref PubMed Scopus (621) Google Scholar), and the structures in the second and third lines show H-bonds possible in different binding conformations of selected inhibitors.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Although protonation state cannot be determined by x-ray crystallography, Crane et al. (49Crane B.R. Arvai A.S. Ghosh D.K. Wu C. Getzoff E.D. Stuehr D.J. Tainer J.A. Science. 1998; 279: 2121-2126Crossref PubMed Scopus (621) Google Scholar) plausibly propose thatl-arginine (p Ka ∼ 12) initially binds as a protonated species; proton donation to heme-bound O2by the proximal, reactive guanidinium nitrogen ofl-arginine then facilitates conversion of O2 to water and the oxo-heme species required for substrate hydroxylation. The product formed, NOH-Arg, has a much lower p Ka(∼7) and is presumed to remain unprotonated, thus facilitating reaction of the second heme-bound O2 as a peroxo rather than as an oxo species (Fig. 6). Because the double bond in NOH-Arg is directed toward the hydroxylated nitrogen (39Boyar A. Marsh R.E. J. Am. Chem. Soc. 1982; 104: 1995-1998Crossref Scopus (57) Google Scholar, 49Crane B.R. Arvai A.S. Ghosh D.K. Wu C. Getzoff E.D. Stuehr D.J. Tainer J.A. Science. 1998; 279: 2121-2126Crossref PubMed Scopus (621) Google Scholar), the distal guanidinium pocket is occupied throughout the catalytic cycle by an –NH2 or =NH2+ group rather than an =NH group; both hydrogens are involved in H-bonds.As shown in Fig. 6, all of the tightly bound l-arginine analogs can be oriented in the active site so as to place –NH2 or =NH2+ in the distal guanidinium nitrogen pocket (e.g. the shorter N ω-alkyl-l-arginines andl-NIO derivatives all have IC50 values indicating binding comparable to or tighter than l-arginine (Table I) and l-thiocitrulline binds to nNOS and iNOS with Ki values of 0.06 and 3.6 μm, respectively (47Frey C. Narayanan K. McMillan K. Spack L. Gross S.S. Masters B.S.S. Griffith O.W. J. Biol. Chem. 1994; 269: 26083-26091Abstract Full Text PDF PubMed Google Scholar)). The presence or absence of charge is apparently of little consequence since both the cationic amidines (i.e. l-NIO and its derivatives) and neutrall-thiocitrulline bind tightly. Similarly, cationicl-arginine and neutral NOH-Arg have similar Km values with iNOS and, more importantly, kcat/Km is only ∼50% higher for l-arginine than for NOH-Arg (55Stuehr D.J. Kwon N.S. Nathan C.F. Griffith O.W. Feldman P.L. Wiseman J. J. Biol. Chem. 1991; 266: 6259-6263Abstract Full Text PDF PubMed Google Scholar). At the typical assay pH of 7.4, l-canavanine possesses a mostly uncharged side chain (p Ka ∼ 7.0) and binds somewhat less tightly than l-arginine despite being isosteric and able to form all of the H-bonds characteristic of the distal guanidinium pocket (Fig. 6). However, presence of oxygen adjacent to N 5 is known to orient the guanidinium double bond into the side chain (39Boyar A. Marsh R.E. J. Am. Chem. Soc. 1982; 104: 1995-1998Crossref Scopus (57) Google Scholar), and consequent loss of the N 5 proton abolishes the H-bond between N 5 and the enzymatic glutamate residue. 4Absence of the N 5 proton and H-bond has been independently described based on an x-ray crystallographic structure of l-canavanine bound to the eNOS oxygenase domain (C. S. Raman, H. Li, P. Martásek, V. Král, B. S. S. Masters, and T. L. Poulos, submitted for publication). The modestly decreased binding affinity of nNOS for l-canavanine is attributed to loss of this H-bond. Comparison of the Ki value of l-canavanine with KmArg (viewed as a “kinetic” binding constant) suggests that the difference in free energy of binding between these species is ∼1.1 kcal/mol. 5Free energy changes were estimated from the van't Hoff equation, ΔG = -2.3 RT log(KEq1/ KEq2). In most cases 1/Ki were used as surrogate KEq values, but comparable results were obtained using 1/Ks or, for l-arginine, 1/Km. It has earlier been shown the KsArg is similar to KmArg (0.7 versus 1.8 μm, respectively) (J. C. Salerno, personal communication). We also limit our analysis to comparison of reasonably isosteric amino acids where differences in binding energy can be confidently attributed to the presence or absence of specific H-bonds. Other classes of inhibitors (e.g. the non-amino acid isothioureas) include compounds that bind with high affinity and yet form few H-bonds (50Raman C.S. Li H. Martásek P. Kral V. Masters B.S.S. Poulos T.L. Cell. 1998; 95: 939-950Abstract Full Text Full Text PDF PubMed Scopus (571) Google Scholar, 56Garvey E.P. Oplinger J.A. Tanoury G.J. Sherman P.A. Fowler M. Marshall S. Harmon M.F. Paith J.E. Furfine E.S. J. Biol. Chem. 1994; 269: 26669-26676Abstract Full Text PDF PubMed Google" @default.
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• W2102657944 title "l-Arginine Binding to Nitric-oxide Synthase" @default.
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