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J Biol Chem, Vol. 274, Issue 36, 25218-25226, September 3, 1999

L-Arginine Binding to Nitric-oxide Synthase
THE ROLE OF H-BONDS TO THE NONREACTIVE GUANIDINIUM NITROGENS^*

Boga Ramesh Babu, Christopher Frey, and Owen W. Griffith

From the Department of Biochemistry, Medical College of Wisconsin, Milwaukee, Wisconsin 53226

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Nitric-oxide synthase (NOS) catalyzes the oxidation of L-arginine to nitric oxide and L-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 the L-arginine-binding site. We report here studies with L-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 of L-canavanine, an analog unable to form hydrogen bonds involving a N⁵-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 K_i values and optical difference spectra to quantitate their affinity relative to L-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.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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-hydroxy-L-arginine (NOH-Arg) is a tightly bound intermediate (1, 2). The enzyme is active as a homodimer, and each monomer is comprised of a heme- and tetrahydrobiopterin-containing oxygenase domain that binds and oxidizes L-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 (3). 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 (1, 2, 4). 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 (5, 6), whereas eNOS produces NO that has an important role in controlling vasorelaxation and blood pressure (7, 8). Nitric oxide derived from iNOS plays both regulatory and cytotoxic roles in the immune response (9, 10). 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 (11) and migraine headache (12), and iNOS is implicated in septic shock (13, 14), inflammatory bowel disease (15), uveitis (16), and arthritis (17, 18). 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 (19-22). 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 the L-arginine-binding site.

In the present studies we have designed and synthesized several novel L-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 (1, 23, 24). We have exploited this difference in size specificity to predictably direct the binding orientation of novel L-arginine analogs. We find that analogs bind poorly if the binding site for the non-reactive guanidinium nitrogen of L-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 PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Materials

Reagents for organic synthesis were obtained from Aldrich and biochemicals were obtained from Sigma, respectively. (6R)-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). N⁵-(1-Iminoethyl)-L-ornithine (L-NIO) (25) and N⁵-acetyl-L-ornithine (26) were prepared by the general methods indicated.

Rat nNOS for most studies was isolated from stably transfected kidney 293 cells as described (27). Bovine eNOS (28, 29) and mouse iNOS (30), 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 (31).

Methods for Synthesis of Arginine Analogs

¹H 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-ornithines-- N⁵-Thioacetyl-L-ornithine (TAO) was synthesized by reaction of an appropriately protected L-ornithine with ethyl dithioacetate. Thus, N-(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 CaCO₃ and 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 (R_F = 0.8). Product-containing fractions were pooled and solvent was evaporated under reduced pressure to give N-(tert-butyloxycarbonyl)-N⁵-(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):  1.75-2.1 (m, 4H), 2.5 (s, 3H), 3.67 (t, 2H) and 4.09 (t, 1H); ¹³C NMR (D₂O):  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.

N⁵-Thiobutyryl-L-ornithine (TBO) was synthesized by reaction of Lawesson's reagent with N⁵-butyryl-L-ornithine, which was obtained by reacting butyryl chloride with protected L-ornithine. Thus, butyryl chloride (2.13 g, 20 mmol) was added to a solution of N-(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-(tert-Butyloxycarbonyl)-N⁵-(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 (R_F = 0.8) and evaporated to give N-(tert-butyloxycarbonyl)-N⁵-(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):  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):  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.

N⁵-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)  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):  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-ornithines-- The following compounds, which are homologs of L-NIO, were synthesized by the general procedure reported previously for N⁵-(1-iminopropyl)-L-ornithine (methyl-L-NIO) (32).

N⁵-(1-Iminobutyl)-L-ornithine (ethyl-L-NIO): m.p. 144-147 °C (dec); ¹H NMR (D₂O):  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):  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).

N⁵-(1-Iminohexyl)-L-ornithine (butyl-L-NIO): m.p. 130-134 °C (dec); ¹H NMR (D₂O):  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):  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-ornithines-- These derivatives were synthesized by dioxane-HCl deprotection of the corresponding N-(tert-butyloxycarbonyl)-N⁵-acyl-L-ornithine tert-butyl esters which were in turn prepared from N-(tert-butyloxycarbonyl)-L-ornithine tert-butyl ester and the appropriate acyl chloride as described above for N-(tert-butyloxycarbonyl)-N⁵-butyryl-L-ornithine tert-butyl ester.

N⁵-Butyryl-L-ornithine: m.p. 65-69 °C; ¹H NMR (D₂O):  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):  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).

N⁵-Hexanoyl-L-ornithine: m.p. 78-80 °C; ¹H NMR (D₂O):  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):  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 Studies

Nitric-oxide Synthase Assays-- Nitric-oxide synthase activity was routinely determined based on the oxidation of oxyhemoglobin to methemoglobin by NO (33). 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 mM NADPH, 20 µM L-arginine, and 5 µM bovine oxyhemoglobin (prepared by reduction with sodium dithionite followed by gel filtration). Formation of methemoglobin was monitored at 401 nm ( = 0.038 µM¹) (33); the reference cuvette contained a similar mixture without enzyme.

IC₅₀ and K_i Determinations-- For determination of most inhibition constants NOS activity was measured by following the conversion of L-[¹⁴C]arginine to L-[¹⁴C]citrulline (32). Reaction mixtures contained in a final volume of 50 µl, 50 mM Na⁺ Hepes buffer, pH 7.4, 100 µM EDTA, 0.2 mM CaCl₂, 10 µg/ml calmodulin, 100 µM dithiothreitol, 50 µM tetrahydrobiopterin, 1.0 µM FAD, 1.0 µM FMN, 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 mM Na⁺ 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 with L-arginine, K_i values were estimated from measured IC₅₀ values using K_i = IC₅₀(K_m^Arg/(K_m^Arg + [Arg])). For purpose of calculation, K_m^Arg values for nNOS, eNOS, and iNOS were estimated as 1.8, 3.6, and 12.5 µM, respectively, based on the present and earlier (32) work.

Optical Difference Spectroscopy-- Interaction of inhibitors with nNOS was determined spectrally using a Shimadzu or Perkin-Elmer dual beam UV/visible spectrophotometer (32). 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.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Binding Orientation of N-Substituted-L-Arginines-- It has previously been shown that N-methyl-L-arginine (L-NMA), the prototypic NOS inhibitor (23), is a pseudo-substrate that is hydroxylated on the methyl-substituted guanidinium nitrogen (34-36). It is also reported that asymmetrical N,N-dimethyl-L-arginine, in which one guanidinium nitrogen is dimethyl-substituted, binds as an effective iNOS inhibitor, but that symmetrical N,N'-dimethyl-L-arginine, in which each guanidinium nitrogen is monosubstituted, is a poorly bound, weak inhibitor (23, 37). We have confirmed the latter results with nNOS (data not shown), and from these observations infer (i) that L-NMA binds with its unsubstituted guanidinium nitrogen in the distal, non-reactive guanidinium nitrogen pocket, (ii) that the binding region for the proximal, reactive guanidinium nitrogen can accommodate both monomethyl- and dimethyl-substitution, and (iii) that the distal pocket cannot accommodate even monomethyl-substitution.

As shown in Table I, N-monoalkyl-L-arginines with ethyl and propyl substituents are also moderately strong inhibitors, whereas the monobutyl derivative is less well bound, especially to iNOS.² These results suggest that the binding region for the guanidinium nitrogen near heme can accommodate substituents extending about 4-5 Å from the guanidinium carbon. Note that the selectivity of N-propyl-L-arginine for inhibition of nNOS has been previously reported by Zhang et al. (38), although our results do not confirm the very large selectivity factors seen in their studies.

Binding Orientation of L-NIO-- In L-NIO one guanidinium -NH₂ group of L-arginine is replaced by -CH₃; the resulting amidine side chain has a pK_a of ~12, assuring its protonation at physiological pH. In principle, L-NIO could bind to NOS with either the =NH₂⁺ or -CH₃ group in the binding region proximal to the heme cofactor. Based on our results with L-NMA homologs and the observation that inhibition by L-NIO is competitive with L-arginine (Fig. 2A), we anticipated that L-NIO homologs containing alkyl groups larger than -CH₃ would be forced to bind with their alkyl groups near heme and their =NH₂⁺ group in the sterically constrained distal guanidinium pocket. Binding affinity would be expected to fall off as the alkyl group became larger than 4-5 carbons. As shown in Table I, these expectations were realized; ethyl-L-NIO with a 3-carbon alkyl group inhibits nearly as well as L-NIO, but butyl-L-NIO with a 5-carbon alkyl group is poorly bound. In earlier studies we showed with all three NOS isoforms that methyl-L-NIO exhibits K_i values that are intermediate between those seen with L-NIO and ethyl-L-NIO (32). This smooth progression in K_i values as alkyl group size increases suggests that, as expected, L-NIO as well as its higher homologs binds with the amidine alkyl group positioned near heme and =NH₂⁺ in the distal guanidinium nitrogen binding pocket.

N⁵-Thioacyl-L-Ornithines as NOS Inhibitors-- L-TAO is a novel L-NIO analog in which =NH₂⁺ is replaced by =S. Higher homologs of L-TAO were also prepared. With respect to alkyl chain length, L-TBO corresponds to ethyl-L-NIO and L-THO corresponds to butyl-L-NIO (Fig. 1). As shown in Fig. 2B, L-TAO-mediated inhibition of nNOS is competitive with respect to L-arginine, suggesting that interaction is with the L-arginine-binding site. The K_i for L-TAO is 34.8 µM, a value ~20-fold higher than that observed in similar studies with L-NIO (Fig. 2A). As shown in Table I, with nNOS the IC₅₀ value for L-TBO is ~1.4-fold higher than that for L-TAO, and inhibition by L-THO is too weak to be usefully quantitated. With iNOS and eNOS, L-TAO and the higher N⁵-thioacyl-L-ornithines inhibit very poorly at accessible concentrations.

View larger version (28K): [in this window] [in a new window]   Fig. 1.   Structures of L-arginine and several novel and known analogs.

View larger version (33K): [in this window] [in a new window]   Fig. 2.   Lineweaver-Burk plot showing that binding of L-NIO (panel A), L-TAO (panel B), and L-canavanine (panel C) to nNOS is competitive with L-arginine. Product formation was determined using the L-[14C]arginine assay (panels A and B) or the hemoglobin NO capture assay (panel C) as described under "Experimental Procedures." The insets in each panel show replots of the data indicating that the Ki values for L-NIO, L-TAO, and L-canavanine are 1.7, 34.8, and 11.1 µM, respectively.

N⁵-Acyl-L-Ornithines as NOS Inhibitors-- N⁵-Acetyl-L-ornithine, N⁵-butyryl-L-ornithine, and N⁵-hexanoyl-L-ornithine are analogs of L-NIO, ethyl-L-NIO, and butyl-L-NIO, respectively, in which the =NH₂⁺ group of the NIO derivatives is replaced by =O (Fig. 1). When tested as NOS inhibitors in studies similar to those shown in Table I, none was an effective inhibitor and all exhibited IC₅₀ values 1 mM (data not shown).

L-Canavanine as a NOS Inhibitor-- L-Canavanine is an L-arginine antagonist in which the -methylene group is replaced by oxygen. The resulting hydroxyguanidine has a pK_a of 7.0 and adopts the imino structure wherein the guanidinium double bond is orientated toward the N⁵ nitrogen (Fig. 1) (39). In consequence, the L-canavanine side chain is not fully protonated at neutral pH and the N⁵ nitrogen does not bear a proton. Although L-canavanine is reported to be an iNOS selective inhibitor (40, 41), we find it inhibits all three isoforms (data for iNOS and eNOS not shown); Fig. 2C shows that inhibition of nNOS is competitive with L-arginine and characterized by a K_i of 11.1 µM at pH 7.4.

To assess the effects of L-canavanine side chain protonation on binding, we determined K_i values for L-canavanine and L-NMA and also determined K_m^Arg as a function of pH (Fig. 3). As shown, affinity for L-arginine, L-canavanine, and L-NMA all decrease with decreasing pH. Although L-canavanine ranges from 24% protonated at pH 7.5 to 90% protonated at pH 6.0, its binding affinity closely tracks that of L-arginine and L-NMA, both of which remain >99.9% protonated over this pH range. At pH values >7.5, nNOS shows decreased affinity for L-canavanine but little change in affinity for L-arginine and L-NMA.

View larger version (17K): [in this window] [in a new window]   Fig. 3.   Plot showing the Ki values for L-canavanine and L-NMA and KmArg as a function of pH. Ki values for L-canavanine and L-NMA were determined at the pH values indicated using the hemoglobin assay to measure NO formation. Ki values and KmArg were determined from Lineweaver-Burk plots of rate data obtained at the pH values indicated.

Possible Direct Interaction between Heme Iron and L-Arginine Analogs-- As isolated, about 80% of nNOS contains high-spin pentavalent heme iron (i.e. 4 bonds to the pyrrole nitrogens of protoporphyrin IX and 1 bond to the sulfur of Cys-415); the remainder of nNOS as isolated contains low-spin, hexavalent heme iron in which an unknown ligand occupies the sixth axial position that is occupied by O₂ during the NOS catalytic cycle (42, 43). When L-arginine, L-NMA, and most amino acid inhibitors bind to NOS, they displace the unknown sixth axial ligand but do not themselves bind to heme iron; the transition to 100% high spin NOS is detected as a type I optical difference spectrum (42-45). As shown in Fig. 4A, L-NIO binds in this manner, giving a type I difference spectrum. In contrast, other inhibitors interact covalently with heme iron, increasing the fraction of low-spin heme and producing a type II optical difference spectrum (46, 47). One such inhibitor is L-thiocitrulline, for which one binding mode includes a bond between its thioureido sulfur and heme iron (47).

View larger version (20K): [in this window] [in a new window]   Fig. 4.   Optical difference spectra of nNOS with L-NIO and L-TAO. Panel A, a sample of nNOS (~2 µM) in 50 mM Tris-HCl buffer, pH 7.4, 10% glycerol, and 0.1 mM EDTA was titrated by adding L-NIO to final concentrations of 10, 20, 30, 40, and 50 µM. The inset shows a double-reciprocal plot of absorbance difference (390-420 nm) versus L-NIO concentration and indicates a spectral dissociation constant (Ks) of 3.6 µM. Panel B, in an experiment similar to that in panel A, nNOS was titrated with L-TAO to final concentrations of 33, 66, 99, and 132 µM. The inset indicates a spectral dissociation constant (Ks) of 57.0 µM.

L-TAO is a structural analog of L-thiocitrulline as well as L-NIO (Fig. 1). Whereas steric constraints force the higher homologs of L-TAO (i.e. L-TBO and L-THO) to bind with their sulfur in the guanidinium-binding region distal to heme, L-TAO might bind in the reverse orientation with -CH₃ in the distal pocket and =S near heme. If so, the sulfur of L-TAO, like the sulfur of L-thiocitrulline, might bind as a sixth axial heme iron ligand and produce a type II optical difference spectrum. However, as shown in Fig. 4B, addition of L-TAO to nNOS produces a type I difference spectrum. The similarity in the spectra for L-TAO and L-NIO, which also has a -CH₃ group in the proximal guanidinium-binding region, is evident. This result is consistent with the view that L-TAO binds with its -CH₃ group rather than =S in the guanidinium-binding region near heme; the distal, non-reactive guanidinium pocket thus favors =S over -CH₃.

L-Citrulline is an extraordinarily weak inhibitor of iNOS (46) and eNOS (K_d > 200 mM) (48) but, when bound, it causes a type II optical difference spectrum (46, 48). As shown in Fig. 5A, L-citrulline also causes a type II difference spectrum when bound to nNOS, a result suggesting that the ureido oxygen is bound to heme iron as a sixth axial ligand. Because N⁵-acetyl-L-ornithine can be viewed as a L-citrulline analog in which -CH₃ replaces -NH₂ (Fig. 1), we determined if exposure of nNOS to high concentrations of N⁵-acetyl-L-ornithine would cause a type II spectrum. As shown in Fig. 5B, type I spectra are obtained, suggesting that the analog binds with its -CH₃ moiety rather than =O near iron. Confirming this result, N⁵-butyryl-L-ornithine also gives a type I difference spectrum (Fig. 5C), and its K_s (0.28 mM) is similar to that for the acetyl derivative (K_s = 0.36 mM).

View larger version (20K): [in this window] [in a new window]   Fig. 5.   Optical difference spectra of nNOS with L-citrulline and N5-acyl-L-ornithines. Panel A, a sample of nNOS (~2 µM) in 50 mM Tris-HCl buffer, pH 7.4, 10% glycerol, and 0.1 mM EDTA was titrated by adding L-citrulline to final concentrations of 2, 2.5, 3.0, and 3.5 mM. The inset shows a double-reciprocal plot of absorbance difference (390-420 nm) versus L-citrulline concentration and indicates a spectral dissociation constant (Ks) of 25 mM. Panel B, in an experiment similar to that in panel A, nNOS was titrated with N5-acetyl-L-ornithine to final concentrations of 0.17, 0.33, 0.66, and 1.33 mM. The inset indicates a spectral dissociation constant (Ks) of 0.36 mM. Panel C, in an experiment similar to that in panel A, nNOS was titrated with N5-butyryl-L-ornithine to final concentrations of 0.25, 0.33, 0.49, 0.88, 1.07, and 1.33 mM. The inset indicates a spectral dissociation constant (Ks) of 0.28 mM.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

High resolution x-ray crystallographic structures for the oxygenase domains of all 3 mammalian NOS isoforms have now been reported in publications (49-51) or at meetings. As expected, the active sites show a high degree of homology, but there are subtle differences (49-52). Consistent with conclusions from substrate specificity (20, 37) and ENDOR (3) studies, the distal, non-reactive guanidinium nitrogen of L-arginine is found to bind >4 Å from heme iron in a sterically constrained pocket³; a conserved enzymatic glutamate residue, identified earlier in mutagenesis studies (53, 54), forms H-bonds to protons on the distal guanidinium nitrogen and the N⁵ 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) (49-51). The present studies further elucidate the importance of these interactions to substrate and inhibitor binding.

View larger version (39K): [in this window] [in a new window]   Fig. 6.   Proposed binding interactions of L-arginine and its analogs with the NOS active site. The top line shows the normal NOS reaction (49), and the structures in the second and third lines show H-bonds possible in different binding conformations of selected inhibitors.

Although protonation state cannot be determined by x-ray crystallography, Crane et al. (49) plausibly propose that L-arginine (pK_a ~ 12) initially binds as a protonated species; proton donation to heme-bound O₂ by the proximal, reactive guanidinium nitrogen of L-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 pK_a (~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 (39, 49), 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-alkyl-L-arginines and L-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 K_i values of 0.06 and 3.6 µM, respectively (47)). The presence or absence of charge is apparently of little consequence since both the cationic amidines (i.e. L-NIO and its derivatives) and neutral L-thiocitrulline bind tightly. Similarly, cationic L-arginine and neutral NOH-Arg have similar K_m values with iNOS and, more importantly, k_cat/K_m is only ~50% higher for L-arginine than for NOH-Arg (55). At the typical assay pH of 7.4, L-canavanine possesses a mostly uncharged side chain (pK_a ~ 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 N⁵ is known to orient the guanidinium double bond into the side chain (39), and consequent loss of the N⁵ proton abolishes the H-bond between N⁵ and the enzymatic glutamate residue.⁴ The modestly decreased binding affinity of nNOS for L-canavanine is attributed to loss of this H-bond. Comparison of the K_i value of L-canavanine with K_m^Arg (viewed as a "kinetic" binding constant) suggests that the difference in free energy of binding between these species is ~1.1 kcal/mol.⁵ This value is much smaller than the average H-bond energy measured in vacuo (3-19 kcal/mol (57)), but is within the range of values typically observed in enzyme-substrate and enzyme-inhibitor interactions where H-bond formation to a ligand is balanced by the loss of H-bonds with water. Such bonds typically contribute 0.5-1.5 kcal/mol of binding energy when uncharged groups are involved and 3-6 kcal/mol when at least one charged group is involved (58). Because the H-bond lost in L-canavanine involves a presumably unprotonated, anionic glutamate residue (49) (Fig. 6), the observed change in binding affinity is ~2 kcal/mol less than predicted, but we are reluctant to invoke specific compensating factors with the limited data available.

Interestingly, at lower pH the K_i values for L-canavanine and L-NMA and K_m^Arg all increase, but there is no improvement in the relative affinity of L-canavanine (Fig. 3). This result suggests that protonation of L-canavanine does not restore the missing H-bond between N⁵ of the inhibitor and the enzymatic glutamate residue but rather, as predicted by Boyar and Marsh (39), L-canavanine protonates on a terminal nitrogen. Furthermore, the similarity in the shapes of the K_i or K_m versus pH profiles for L-canavanine, L-NMA, and L-arginine at low pH suggests that the enzymatic glutamate residue does not protonate even at pH 6 to restore an H-bond with N⁵ of L-canavanine.⁶ The apparent loss of affinity for L-canavanine at pH > 8 is distinct from what is seen with L-NMA and L-arginine and is not presently understood in terms of specific interactions.

Although earlier mutagenesis studies identified Glu-371 of iNOS (53) and Glu-361 of eNOS (54) as important for L-arginine binding, those results could not be unambiguously interpreted until x-ray crystallographic studies established that H-bonds to that residue stabilize the -amino, distal guanidinium and N⁵-nitrogens of L-arginine (49-51). For both iNOS and eNOS, replacement of the glutamate residue with alanine (iNOS) or leucine (eNOS) completely abolished L-arginine binding (53, 54), a result indicating the critical importance of the H-bonds made to that residue.⁷ In the present studies we have modified the substrate rather than the enzyme and have been able to selectively probe the importance of specific H-bonds. Our results suggest that H-bonds to the distal guanidinium nitrogen or to the N⁵-nitrogen are likely to be important in establishing guanidinium group orientation, but they are relatively weak (<2 kcal) and therefore not individually essential to binding per se. Thus, by taking advantage of the unique ability of the proximal, reactive guanidinium nitrogen-binding site to accommodate n-alkyl substituents as large as 3-4 carbons, we have designed L-arginine analogs that can bind only if the distal guanidinium pocket is occupied by groups other than -NH₂ or =NH₂⁺. We have shown that some of those analogs bind despite being unable to form H-bonds to the enzymatic glutamate and tryptophan residues identified by x-ray crystallography (49-51). Because we have spectral data only with nNOS, we limit our detailed analysis to that isoform and then comment briefly on differences with iNOS and eNOS.

L-TAO is a weak competitive inhibitor of nNOS; its K_i is 34.8 µM, 21-fold higher than K_m^Arg. As shown in Fig. 6, L-TAO might bind in either or both of two conformations. Conformation B, in which -CH₃ occupies the distal guanidinium nitrogen pocket and =S occupies the region near heme, is analogous to the preferred binding mode of L-thiocitrulline (47, 49, 59). Because L-thiocitrulline (K_i = 0.06 µM (47)) binds much more tightly than L-arginine (K_m = 1.8 µM), L-NIO (K_i = 1.7 µM (this work and Ref. 32)), or L-citrulline (K_s ~ 25 mM (this work)), it is clear that occupancy of the region near heme by =S rather than -NH₂, -CH₃, or =O is energetically favorable. That said, optical difference spectroscopy with L-TAO (Fig. 4B) provides no evidence for the sulfur-heme interaction seen with L-thiocitrulline. Although that finding argues against binding in conformation B, we note that sulfur-heme bonds are seen for only 10-20% of the L-thiocitrulline bound to nNOS and are not seen with L-homothiocitrulline, which is nonetheless thought to bind analogously (47, 59). It is thus possible that some L-TAO binds in conformation B but with its sulfur atom sufficiently far from heme iron to prevent covalent interaction. If that is the mode of binding, the loss of binding energy attributable to placing -CH₃ rather than -NH₂ in the distal guanidinium nitrogen pocket can be calculated from the difference in K_i values between L-TAO and L-thiocitrulline (34.8 µM versus 0.06 µM (47)). The G difference is ~3.8 kcal/mol, somewhat lower than the 6-12 kcal/mol expected for the loss of two H-bonds involving charged groups (Fig. 6).

Conformation A for L-TAO (Fig. 6) places =S in the distal guanidinium nitrogen pocket and -CH₃ near heme; in this conformation L-TAO can be viewed as an analog of L-NIO in which =S replaces =NH₂⁺. If the observed loss of binding affinity is fully attributed to this structural difference, then comparison of K_i values (34.8 µM versus 1.7 µM) suggests a G difference of ~1.8 kcal/mol. This is again a surprisingly small value considering two H-bonds involving charged groups are lost (i.e. the enzymatic glutamate is thought to be ionized and the amidinium group of L-NIO is certainly cationic). Thus, independent of binding conformation, the L-TAO results suggest that the H-bonds involving residues in the distal guanidinium pocket are relatively weak.

For the higher homologs of L-TAO, there is no ambiguity in binding conformation; the size of the alkyl substituent precludes conformer B-type binding. Calculating K_i values from the data in Table I, the affinity of nNOS for L-TBO (K_i = 31 µM) compared with ethyl-L-NIO (K_i = 5.2 µM (Table I) or 5.3 µM (32)) suggests that binding energy decreased ~1.05 kcal/mol in L-TBO. This value is again substantially smaller than would be expected for loss of two H-bonds involving charged groups. However, the relatively close similarity between this value and ~1.8 kcal/mol estimated for L-TAO binding in conformation A supports the view that conformation A does, in fact, best represent L-TAO binding. Although we cannot rigorously exclude the possibility that some L-TAO binds in conformation B, the simplest interpretation of our results is that it is energetically more favorable to place =S rather than -CH₃ in the distal guanidinium binding pocket. This selectivity is unlikely to be attributable to the small difference in van der Waals radii between the groups (1.85 Å for =S versus 2.0 Å for -CH₃ (60)), but may reflect the much greater polarizability of sulfur and its ability to form bonds based on dispersion forces.

Electron spin resonance studies by Salerno et al. (61) showed that L-citrulline binds as a sixth axial ligand to heme iron in nNOS; the major interaction is through =O but a minority species shows iron bonded to the -NH₂ group. That result, coupled with the poor overall affinity for L-citrulline (K_s ~ 25 mM (Fig. 5A)), suggests (i) that the binding energy for L-citrulline bound with =O near heme is similar to the binding energy with =O in the distal guanidinium binding pocket, and (ii) that =O is very poorly bound at either site. N⁵-Acetyl-L-ornithine is a L-citrulline analog and, like L-citrulline, is weakly bound. However, N⁵-acetyl-L-ornithine binding apparently favors =O in the distal pocket since we observed a type I optical difference spectrum with the compound (Fig. 5B). Confirming that view, N⁵-butyryl-L-ornithine, which for steric reasons must bind with =O in the distal pocket, also gives a type I spectrum (Fig. 5C), and the K_s values for the acetyl and butyryl compounds are comparable (0.37 and 0.28 mM, respectively). Comparison of these values to the K_i values for L-NIO and ethyl-L-NIO (or the K_s for L-NIO) indicates that binding affinity is decreased by 2.7-3.2 kcal/mol (N⁵-acetyl-L-ornithine versus L-NIO) or 2.4 kcal/mol (N⁵-butyryl-L-ornithine versus ethyl-L-NIO). These are again unexpectedly low values considering two H-bonds involving charged groups were lost. Taken together with the similar findings for the corresponding sulfur derivatives (TAO and TBO), our results suggest that the two H-bonds involving the distal guanidinium nitrogen of L-arginine are much weaker (0.9-1.5 kcal/mol each) than would have been predicted for such bonds (3-6 kcal/mol each). Such relative weakness may reflect an unfavorable H-bond length or very favorable interactions of glutamate and tryptophan with water or other residues that are then lost on binding L-arginine or its analogs. Although bond lengths are not yet reported for nNOS, the corresponding bonds in eNOS are 3.19 Å (Glu-363^{. . . . .} H₂⁺N=) and 2.88 Å (Trp-358^{. . . . .} H₂⁺N=)⁸; at least the former is too long for maximum strength.

Taken together our results indicate that the distal guanidinium nitrogen binding pocket of nNOS has highest affinity for -NH₂ and =NH₂⁺, moderate affinity for =S, substantially less affinity for =O, and very little affinity for -CH₃. The binding region near heme (i.e. the proximal guanidinium nitrogen site) has highest affinity for =S (and -S-alkyl (45, 62)), very little affinity for =O, and moderate affinity for alkyl, -NH-alkyl, and -NH₂ or =NH₂⁺ (approximately in that order). The results shown in Table I suggest iNOS and eNOS differ from nNOS in several respects. Most dramatically, iNOS is less able to bind N-alkyl groups larger than ethyl near heme, and eNOS and, to a lesser extent, iNOS do not accommodate =S in the distal guanidinium pocket as well as nNOS does. Fan et al. (52) similarly concluded from resonance Raman studies that the nNOS L-arginine-binding site is more open than that of iNOS and eNOS (52). Finally, we note that most NOS inhibitors designed or discovered to date bind competitively with L-arginine and are therefore presumed to occupy at least part of the L-arginine-binding site. With the exception of certain aromatic compounds (e.g. 7-nitroindazole), almost all such inhibitors include Structure I as a structural motif. The present results strongly suggest that the =NH₂⁺ moiety of that motif is bound in the distal guanidinium pocket. Although that binding region will accommodate other groups as long as they are not larger than =NH₂⁺, even the best of the surrogates (e.g. =S for nNOS) is bound with substantially less affinity. We conclude that inhibitors targeting the guanidinium region of the L-arginine-binding site should include a guanidine, amidine, thiourea, or isothiourea motif.

	ACKNOWLEDGEMENTS

We thank Michael A. Hayward for expert technical assistance and Drs. K. Pritchard, L. J. Roman, and B. S. S. Masters for generous gifts of purified NOS isoforms. We thank Dr. C. S. Raman for providing detailed information on the eNOS structure and helpful discussions on L-canavanine binding.

	FOOTNOTES

^* This work was supported in part by National Institutes of Health Grant DK48423.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: Dept. of Biochemistry, Medical College of Wisconsin, Milwaukee, WI 53226. Tel.: 414-456-8435; Fax: 414-456-6510; E-mail: griffith@mcw.edu.

² Interestingly, N-alkyl-L-arginines with n-alkyl substituents of 12-16 carbons are also potent inhibitors. Such compounds do not bind competitively with L-arginine and do not cause type I or type II optical difference spectra (B. R. Babu and O. W. Griffith, unpublished). The mechanism by which long chain N-alkyl-L-arginines inhibit will be the subject of a separate report.

³ In eNOS the distance between heme iron and the distal guanidinium and the N⁵ nitrogens are 4.7 and 4.9 Å, respectively (Ref. 50, and C. S. Raman, personal communication).

⁴ Absence of the N⁵ 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).

⁵ Free energy changes were estimated from the van't Hoff equation, G = -2.3 RT log(K_Eq¹/K_Eq²). In most cases 1/K_i were used as surrogate K_Eq values, but comparable results were obtained using 1/K_s or, for L-arginine, 1/K_m. It has earlier been shown the K_s^Arg is similar to K_m^Arg (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 (50, 56). The affinity of NOS for such compounds depends on interactions not present in the amino acids studied here, and differences in binding affinities cannot therefore be attributed to gain or loss of specific H-bonds.

⁶ That is, if the glutamate residue were to protonate as the pH decreased from 7.5 to 6.0, then the missing H-bond could be restored, forming between the glutamate proton and the unprotonated N⁵ of L-canavanine; affinity for L-canavanine would be expected to improve at lower pH relative to L-NMA or L-arginine. This is not observed. Similarly, if the glutamate residue were protonated even at neutral pH, the missing proton on L-canavanine would not affect the number of H-bonds stabilizing binding, and the affinity for L-canavanine would be expected to match that for L-arginine. It does not.

⁷ In eNOS replacement of Glu-361 with glutamine also abolished activity (54). Since glutamine could form as many H-bonds as glutamate, we tentatively attribute loss of activity in the E361Q mutant to a conformational misalignment of the glutamine residue. Loss of charge interaction with the protonated guanidinium group of L-arginine is an alternative explanation favored by P-F. Chen et al. (54), but, as discussed above, native NOS binds both cationic and neutral L-arginine analogs and we think this explanation is less likely. It would be interesting to know if the E361Q mutant binds N-nitro-L-arginine, a tightly bound, but uncharged inhibitor.

⁸ C. S. Raman, personal communication.

	ABBREVIATIONS

The abbreviations used are: NOS, nitric-oxide synthase; nNOS, neuronal NOS; eNOS, endothelial NOS; iNOS, inducible NOS; NO, nitric oxide; NOH-Arg, N-hydroxy-L-arginine; L-NMA, N-methyl-L-arginine; L-NIO, N⁵-(1-iminoethyl)-L-ornithine; ethyl-L-NIO, N⁵-(1-iminobutyl)-L-ornithine; butyl-L-NIO, N⁵-(1-iminohexyl)-L-ornithine; TAO, N⁵-thioacetyl-L-ornithine; L-TBO, N⁵-thiobutyryl-L-ornithine; L-THO, N⁵-thiohexanoyl-L-ornithine; K_s, dissociation constant determined from spectral studies; EI, electron impact; FAB-MS, fast atom bombardment-mass spectroscopy.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES