Relationship between the structure of guanidines and N-hydroxyguanidines, their binding to inducible nitric oxide synthase (iNOS) and their iNOS-catalysed oxidation to NO ` David Lefevre-Groboillot1,2, Jean-Luc Boucher1, Dennis J Stuehr2 and Daniel Mansuy1 ´ Laboratoire de Chimie et Biochimie Pharmacologiques et Toxicologiques, Universite Paris 5, France Department of Immunology, Lerner Research Institute, Cleveland, OH, USA Keywords binding kinetic; guanidines N-hydroxyguanidines; nitric oxide synthase; UV ⁄ Vis difference spectroscopy Correspondence J-L Boucher, Laboratoire de Chimie et Biochimie Pharmacologiques et Toxicologiques, UMR 8601 CNRS, ´ ` Universite Paris 5, 45 rue des Saints-Peres, 75270 Paris Cedex 06, France Fax: +33 42 86 83 87 Tel: +33 42 86 21 91 E-mail: boucher@biomedicale.univ-paris5.fr (Received 18 February 2005, revised 20 April 2005, accepted 25 April 2005) doi:10.1111/j.1742-4658.2005.04736.x The binding of several alkyl- and aryl-guanidines and N-hydroxyguanidines to the oxygenase domain of inducible NO-synthase (iNOSoxy) was studied by UV ⁄ Vis difference spectroscopy In a very general manner, monosubstituted guanidines exhibited affinities for iNOSoxy that were very close to those of the corresponding N-hydroxyguanidines The highest affinities were observed for the natural substrates, l-arginine and Nx-hydroxyl-arginine (Kd at the lm level) The deletion of either the CO2H or the NH2 function of their amino acid moiety led to dramatic decreases in the affinity However, alkylguanidines with a relatively small alkyl chain exhibited interesting affinities, the best being observed for a butyl chain (Kd ¼ 20 lm) Arylguanidines also bound to iNOSoxy, however, with lower affinities (Kd > 250 lm) Many N-alkyl- and N-aryl-N¢-hydroxyguanidines are oxidized by iNOS with formation of NO, whereas only few alkylguanidines led to significant production of NO under identical conditions, and all the arylguanidines tested to date were unable to lead to the production of NO The kcat values of NO production from the oxidation by iNOS of the studied N-hydroxyguanidines were found to vary independently of their affinity for the protein The kcat values determined for the two-step oxidation of alkylguanidines to NO were not clearly related to the Kd of these substrates toward iNOSoxy However, there is a qualitative relationship between these kcat values and the apparent rate constants of dissociation of the complex between iNOSoxy and the corresponding N-alkyl-N¢-hydroxyguanidine (koff app ) that were determined by stopped-flow UV ⁄ Vis spectroscopy These data indicate that a key factor for efficient oxidation of a guanidine by iNOS to NO is the ability of the corresponding N-hydroxyguanidine to bind to the active site without being too rapidly released before its further oxidation This explains why 4,4,4-trifluorobutylguanidine is so far the best non-a-amino acid guanidine substrate of iNOS with formation of NO, because the koff app of the corresponding N-hydroxyguanidine is particularly low This suggests that the rational design of guanidines as new NO donors Abbreviations BH4, (6R)-5,6,7,8-tetrahydro-L-biopterin; BuGua, n-butylguanidine; BuNOHG, N-(n-butyl)-N ¢-hydroxyguanidine; BzNOHG, N-benzyl-N ¢hydroxyguanidine; ClPhNOHG, N-(4-chlorophenyl)-N ¢-hydroxyguanidine; FPhGua, 4-fluorophenylguanidine; FPhNOHG, N-(4-fluorophenyl)N ¢-hydroxyguanidine; HexGua, n-hexylguanidine; HexNOHG, N-(n-hexyl)-N ¢-hydroxyguanidine; homo-L-Arg, homo-L-arginine; homo-NOHA, N x-hydroxy-homo-L-arginine; HS, high spin; ImH, imidazole; L-Arg, L-arginine; LS, low spin; NOHA, N x-hydroxy-L-Arginine; Nor-L-Arg, nor-LArginine; NOHAgma, N x-hydroxyagmatine; NOHGPA, N x-hydroxyguanidinopentanoic acid; NOS, nitric oxide synthase; NOSoxy, oxygenase domain of NOS; PentylGua, n-pentylguanidine; PentylNOHG, N-(n-pentyl)-N ¢-hydroxyguanidine; ProGua, n-propylguanidine; ProNOHG, N-(n-propyl)-N ¢-hydroxyguanidine; TFBGua, 4,4,4-trifluorobutylguanidine; TFBNOHG, N-(4,4,4-triuorobutyl)-N Â-hydroxyguanidine 3172 FEBS Journal 272 (2005) 31723183 ê 2005 FEBS ` D Lefevre-Groboillot et al Substrate specificity of iNOS upon in situ oxidation by NOSs should take into account both thermodynamic and kinetic characteristics of the interaction of the protein not only with the guanidine but also with the corresponding N-hydroxyguanidine Nitric oxide synthases (NOS) catalyse the oxidation of l-arginine (l-Arg) into l-citrulline and NO, with the intermediate formation of Nx-hydroxy-l-arginine (NOHA) [1–3] This reaction ideally consumes 1.5 mol of NADPH and mol of O2 It occurs in the homodimeric N-terminal domain of the protein called NOS oxygenase domain (NOSoxy) that contains two cofactors per monomer, the heme (iron-protoporphyrin IX) and (6R)-5,6,7,8-tetrahydro-l-biopterin (BH4) Electrons from NADPH are provided to heme by flanking C-terminal reductase domains NOSs are heme-thiolate monooxygenases comparable with cytochrome P450 Whereas proteins of the cytochrome P450 family are known to be able to bind and oxidize a very large number of compounds of various structures, until recently NOSs were only known to be able to oxidize l-Arg and a very small number of its close a-amino acid analogues Recent reports have shown that NOSs are able to produce NO from the oxidation of many non-a-amino acid monosubstituted N-hydroxyguanidines, including N-alkyl-N¢-hydroxyguanidines and N-aryl-N¢-hydroxyguanidines, provided that the alkyl or aryl substituent is neither too small nor too bulky [4–7] NOS-catalysed oxidation of some of these compounds showed kcat values as high as 80% that obtained with NOHA, and some proved to be selective for one of the three isoforms vs the others [5,7] More recently, NO production has also been observed from the oxidation of several non-a-amino acid alkylguanidines by purified iNOS or by activated mouse macrophages, opening the way to the design of stable exogenous NOS substrates of pharmacological interest [8,9] Apart from some equilibrium and kinetic constants related to the binding of l-Arg [10–14] and NOHA [10], nothing is known about the thermodynamics and kinetics of the binding of guanidines and N-hydroxyguanidines to iNOS Removal of the a-amino or a-carboxylate moiety of l-Arg has important effects on the ability of the resulting compounds to affect the heme iron spin equilibrium, and to trigger NADPH consumption and NO production [14–16] Interestingly, it has been shown that several binding modes exist for N-hydroxyguanidines in the heme pocket of NOSs [17– 20] Also, the fact that isoform-selective substrates for NOS [5,7,9] were characterized is striking, given the high level of similarity between the crystal structures of the oxygenase domains of the three isoforms [18,19,21] FEBS Journal 272 (2005) 3172–3183 ª 2005 FEBS This study was undertaken to determine structural factors that are important for a guanidine or N-hydroxyguanidine to be well recognized by the NOS active site, and to be efficiently oxidized with NO formation For that purpose, the dissociation constants of several complexes of the oxygenase domain of iNOS (iNOSoxy) with various alkyl- and aryl-guanidines and N-hydroxyguanidines were determined by UV ⁄ Vis difference spectroscopy, according to a previously described technique [22] The kinetics of the binding of some of these substrates to iNOSoxy was also studied by UV ⁄ Vis spectroscopy using stopped-flow techniques [23,24] The corresponding thermodynamic and kinetic binding constants were then compared with the kinetic constants of NO formation from iNOS-catalysed oxidation of guanidine and N-hydroxyguanidine substrates Our results suggest that a key factor in the efficient oxidation of a guanidine to NO by iNOS could be the ability of the corresponding N-hydroxyguanidine to bind to the active site without being too rapidly released before its further oxidation Our results may help in the further rational design of guanidines as new NO precursors Results Study of the binding of guanidines and N-hydroxyguanidines to iNOSoxy by UV ⁄ Vis difference spectroscopy Purified recombinant iNOSoxy showed a wide Soret band with a maximum absorption wavelength around 400 nm, indicating that the heme-iron existed in equilibrium between a hexacoordinated low-spin (LS) state and a pentacoordinated high-spin (HS) state, the major fraction being in the HS state As previously described, addition of l-Arg leads to conversion of the minor population of heme centres being in the LS state into the HS state and to the appearance of a difference spectrum [22,25–27] However, the intensity of this difference spectrum is small, because the spin state of the major fraction of the protein is not affected Imidazole (ImH) was thus used to completely convert iNOSoxy into a LS state iNOSoxy–Fe(III)–ImH complex allowing one to more easily follow the binding of guanidines or N-hydroxyguanidines to the iNOSoxy substrate binding site [12,22,28] iNOSoxy (1 lm) in the presence of 400 lm ImH was first titrated with l-Arg A difference spectrum displaying a peak at 392 nm and a 3173 ` D Lefevre-Groboillot et al Substrate specificity of iNOS Fig Difference spectrum obtained upon addition of increasing concentrations of L-Arg to iNOSoxy in the presence of ImH iNOSoxy and ImH concentrations were and 400 lM, respectively (Inset) Plot of ⁄ DA vs ⁄ [L-Arg] trough at 430 nm (Fig 1) resulting from the conversion of the LS NOS–Fe(III)–ImH complex to the HS NOS–Fe(III)–l-Arg complex was observed Inhibition of the binding of l-Arg to iNOSoxy by ImH, and of the iNOS-catalysed conversion of l-Arg into l-citrulline has previously been shown to be competitive [11] Equation (1) was thus used to calculate corrected equilibrium constants, Kd, for the iNOSoxy–substrate complexes from apparent constants Kapp [12–14,23,24] Kapp =Kd ¼ ỵ ẵImH=KImH 1ị With the ImH concentration used in this study (400 lm), Eqn (1) became Kapp ¼ 8:7Kd ð10 Þ Variations in the amplitudes of the difference spectra with the concentrations of l-Arg were in agreement with a single binding site model (see Experimental procedures) and Kapp value of 26 ± lm was found for the apparent equilibrium constant for the dissociation of the iNOSoxy–l-Arg complex, in good agreement with a previously reported value (28 ± lm) obtained with the same ImH concentration [25] At the end of the titration, the absolute spectrum of the iNOSoxy solution containing 400 lm ImH and mm l-Arg showed a maximum absorption wavelength at 395 ± nm (not shown) Similar titrations of iNOSoxy in the presence of 400 lm ImH were then performed with a large number of guanidines and N-hydroxyguanidines previously 3174 evaluated as iNOS substrates [4–9,29] The positions of the peaks and troughs of the difference spectra observed during these titrations were similar to those observed when l-Arg was used (Fig 1) Variation in the amplitude of the observed difference spectra with the concentration of the studied guanidines or N-hydroxyguanidines was always in reasonable agreement with a single binding site model The apparent equilibrium constants derived from these experiments are shown in Table NOHA was found to bind to iNOS with a Kapp value slightly lower than that of l-Arg (18 ± lm, Table 1) Homo-L-Arg and homo-NOHA, the l-Arg and NOHA analogues bearing one extra methylene group in the alkyl side-chain, were found to bind to iNOSoxy with higher Kapp values than l-Arg (80 ± 13 and 150 ± 40 lm, respectively) Finally, a much higher Kapp value (2.4 mm) was found for nor-l-Arg, the analogue bearing one methylene fewer than l-Arg Removal of either the a-COOH or the a-NH2 group of NOHA led to a dramatic decrease in the affinity of the resulting compounds, the Kapp values measured for Nx-hydroxyagmatine (NOHAgma), and Nx-hydroxyguanidino-pentanoic acid (NOHGPA), being > mm (2 and > mm, respectively; Table 1) However, the simultaneous removal of both the a-NH2 and a-COOH functions of NOHA led to N-(n-butyl)-N¢-hydroxyguanidine (BuNOHG), which showed a much lower Kapp value of 160 ± 40 lm (Table 1) Replacement of the terminal CH3 group of the n-butyl chain by a CF3 group, leading to N-(4,4,4-trifluorobutyl)-N¢-hydroxyguanidine (TFBNOHG), resulted in a sixfold increase in the Kapp value Shorter nonfunctionalized analogues N-(n-propyl)-N¢-hydroxyguanidine (ProNOHG) and longer ones N-(n-pentyl)-N¢-hydroxyguanidine (PentylNOHG) and N-(n-hexyl)-N¢-hydroxyguanidine (HexNOHG) showed higher Kapp values than the N-(n-butyl) compound (270, 900 and >1000 lm, respectively) Finally, N-benzyl-N¢-hydroxyguanidine (BzNOHG) and the three para-substituted aryl-derivatives N-(4-fluoro-, 4-methyl- and 4-chlorophenyl)-N¢hydroxyguanidines (FPhNOHG, TolNOHG and ClPhNOHG), showed Kapp values > mm A study of the binding of the corresponding nonfunctionalized alkylguanidines to iNOSoxy led to very similar conclusions In the studied series, the alkylguanidine exhibiting the highest affinity for iNOS was n-butylguanidine (BuGua), with a Kapp value of 140 ± 20 lm (Table 1) Trifluorination of the terminal methyl group of the n-butyl chain, leading to 4,4,4-trifluorobutylguanidine (TFBGua), increased the Kapp value by 10-fold The longer nonsubstituted n-pentyland n-hexylguanidines (PentylGua and HexGua) also FEBS Journal 272 (2005) 3172–3183 ª 2005 FEBS ` D Lefevre-Groboillot et al Substrate specificity of iNOS Table Apparent equilibrium constants (Kapp) for the binding of N-hydroxyguanidines R-NH-C(¼ NOH)-NH2 and guanidines R-NH-C(¼ NH2)NH2 to iNOSoxy Titrations were performed by UV ⁄ Vis difference spectroscopy in the presence of 25 lM BH4, mM dithiothreitol and 400 lM ImH Kapp values were calculated as described in Experimental procedures Values ± SD from three different experiments n.d., not determined R N-Hydroxyguanidines Compound Kapp HO2C-CH(NH2)-(CH2)3HO2C-CH(NH2)-(CH2)4HO2C-CH(NH2)-(CH2)2HO2C-(CH2)4H2N-(CH2)4CH3(CH2)2 CH3(CH2)3 CF3(CH2)3CH3(CH2)4 CH3(CH2)5 4-F-C6H44-CH3-C6H44-Cl-C6H4C6H4-CH2- NOHA Homo-NOHA – NOHGPA NOHGAgma ProNOHG BuNOHG TFBNOHG PentylNOHG HexNOHG FPhNOHG TolNOHG ClPhNOHG BzNOHG 18 ± lM 150 ± 40 lM n.d > mM 2.0 ± 0.7 mM 270 ± 50 lM 160 ± 40 lM 0.9 ± 0.2 mM 0.7 ± 0.3 mM > mM 2.4 ± 0.1 mM > mM 3.0 ± 0.5 mM > mM showed higher Kapp values (600 lm and > mm for the n-pentyl and n-hexyl derivatives, respectively) The shorter n-propylguanidine (ProGua) showed a Kapp value similar to that found for BuGua Finally, the arylguanidines 4-fluorophenyl- and 4-methylphenylguanidines (FPhGua and TolGua) interacted with iNOS with Kapp values>2 mm Relationship between the equilibrium constants measured for the binding of guanidines and N-hydroxyguanidines to iNOSoxy and the kinetic constants measured for their iNOS-catalysed oxidation to NO In previous studies, we have identified some N-alkyland N-aryl-N¢-hydroxyguanidines, and alkylguanidines as NO donors following their oxidation catalysed by Guanidines Compound Kapp L-Arg 26 ± lM 80 ± 13 lM 2.4 ± 1.0 mM n.d n.d 140 ± 20 lM 140 ± 20 lM 1.5 ± 0.4 mM 0.6 ± 0.1 mM > mM 2.0 ± 0.1 mM 6.0 ± 1.5 mM n.d n.d Homo-L-Arg Nor-L-Arg – – ProGua BuGua TFBGua PentylGua HexGua FPhGua TolGua – – iNOS containing all its cofactors [4,5,8,9] Table gives the Km and kcat values measured for the oxidation of seven N-hydroxyguanidines leading to the highest production of NO in the presence of iNOS, together with Km and kcat values for the oxidation of the corresponding guanidines [4,5,8,9] The seven N-hydroxyguanidines NOHA, homo-NOHA, BuNOHG, TFBNOHG, PentylNOHG, FPhNOHG and TolNOHG were oxidized with formation of NO with similar high kcat values ranging from 58 to 100% of that found for NOHA They showed widespread Km ⁄ Kd ratios, generally >1 and that varied from  to  20 (Table 2) In that series, the kcat value for the production of NO from the oxidation of the N-hydroxyguanidines varied by less than a factor 2, whereas the kcat value for the production of NO from the oxidation of the guanidines varied a great deal from to 100% of the kcat value obtained Table Kinetic constants for the formation of NO from the oxidation of guanidines and N-hydroxyguanidines by recombinant iNOS See Table for the structure of compounds Km and kcat values are taken from previous publications [4,5,8,9,29] kcat values are expressed per NOS dimer The corrected dissociation equilibrium constants (Kd) for the binding of guanidines and N-hydroxyguanidines to iNOSoxy were obtained by dividing Kapp values (taken from Table 1) by 8.7 Compounds Km (lM) N-Hydroxy guanidine NOHA ⁄ L-Arg Homo-NOHA ⁄ Homo-L-Arg BuNOHG ⁄ BuGua TFBNOHG ⁄ TFBGua PentylNOHG ⁄ PentylGua FPhNOHG ⁄ FPhGua TolNOHG ⁄ TolGua 40 146 55 840 310 300 1100 a ± ± ± ± ± ± ± 10 21 10 100 50 40 300 Guanidine kcat (min)1) N-Hydroxy guanidine 33 45 275 250 – – 480 410 320 780 280 350 295 ± ± ± ± ± 10 50 100 ± ± ± ± ± ± ± 60 50 50 100 50 80 50 Guanidine Km ⁄ Kd N-Hydroxy guanidine Ref 400 ± 215 ± 23 ± 220 ± 60 ± < 2a < 2a 19.3 8.5 3.0 8.1 3.8 1.1 < 2.0 [4,8,9] [29] [4,8] [8,9] [4,9] [5,8,9] [5,9] 50 50 50 15 The rates of the production of NO from the oxidation by iNOS of mM FPhGua or TolGua were lower than min)1 FEBS Journal 272 (2005) 3172–3183 ª 2005 FEBS 3175 ` D Lefevre-Groboillot et al with l-Arg, with the order l-Arg > homo-lArg  TFBGua > PentylGua > BuGua > FPhGua >  TolGua (Table 2) The Km and kcat values calculated for NO formation from iNOS-catalysed oxidation of guanidines are complex parameters as they correspond to a two-step reaction with intermediate formation of N-hydroxyguanidines The data are difficult to correlate with kinetic or thermodynamic constants clearly describing individual reactions, such as Kd (or Kapp) The situation should be less complex for Km and kcat for NO formation from N-hydroxyguanidines that are more closely related to a one-step enzymatic reaction It is actually well known that, for enzymes having high kcat values, the Km values can be markedly higher than the Kd values, as indicated by the classical relation given here [30] Absorbance Substrate specificity of iNOS Km ¼ Kd ỵ kcat =kon B A 392 nm 0.263 0.213 0.163 0.113 0.063 350 370 390 410 430 450 470 490 Wavelength (nm) or 0.33 430 nm 0.32 Km =Kd ẳ ỵ kcat =koff 0.31 Absorbance This equation implies that Km ⁄ Kd will increase as kcat increases and koff decreases Because the kcat value found for these seven N-hydroxyguanidines varied by less than a factor 2, it was tempting to investigate a possible relationship between Km ⁄ Kd and koff The following experiments were performed as a first approach to find the variation in koff as a function of the iNOS substrate structure 430 nm 0.313 0.3 0.29 0.28 0.27 0.26 392 nm Kinetics of the binding of guanidines and N-hydroxyguanidines to iNOSoxy measured by stopped-flow UV ⁄ Vis spectroscopy An iNOSoxy solution containing 400 lm ImH was rapidly mixed with a solution of the studied ligand containing the same concentration of ImH Postmixing ligand concentrations corresponded to pseudo-first-order conditions Absorption variations were monitored at 430 and 392 nm (Fig 2), allowing one to follow, respectively, the disappearance of the NOS–Fe(III)–ImH complex and the appearance of the high-spin NOS–Fe(III) species The calculated kinetic constants kobs were plotted against the ligand concentration and satisfactorily fitted with a linear function kobs ẳ koff app ỵ koff app ẵL where L is the guanidine or N-hydroxyguanidine used (Fig 3), in agreement with a competitive model for the interaction between ImH and the studied guanidine or N-hydroxyguanidine [23,24,28] It has previously been 3176 0.25 0.5 1.5 Time (s) Fig Spectral transitions observed as a function of time upon the fast addition of BuNOHG to iNOSoxy in the presence of ImH ImH concentration was 400 lM Final heme and BuNOHG concentrations were lM and mM, respectively (A) Rapid-scanning stopped-flow spectra recorded during the reaction (B) Cross-section of (A) variation in absorbance at 430 and 392 nm as a function of time shown that displacement of ImH from the NOS hemeiron by l-Arg or its analogues is a two-step process [23,28] and might involve an intermediate and transient ternary complex between the protein, ImH and the l-Arg analogue [23] The konapp and koff app values are thus apparent association and dissociation rate constants of the guanidine or N-hydroxyguanidine with the protein in the presence of 400 lm ImH Three guanidines and the corresponding N-hydroxyguanidines were studied l-Arg and NOHA were used FEBS Journal 272 (2005) 3172–3183 ª 2005 FEBS ` D Lefevre-Groboillot et al Substrate specificity of iNOS as reference compounds and two pairs of non-a-amino acid compounds, BuGua ⁄ BuNOHG and TFBGua ⁄ TFBNOHG were also studied The determined values of konapp and koff app are reported in Table In the studied range of concentrations, the kobs values were higher for a guanidine than for the corresponding Nhydroxyguanidine (Fig 3) The konapp values for the guanidines were found to be 5–10 higher than those for the corresponding N-hydroxyguanidines, and the koff app values for the guanidines were 25–60 times higher than those for the corresponding N-hydroxyguanidines (Table 3) The konapp values for the nona-amino acid guanidines BuGua and TFBGua were found to be 7- and 25-fold lower than that for l-Arg, and those for the non-a-amino acid N-hydroxyguanidines BuNOHG and TFBNOHG were 3- and 12-fold lower than that for NOHA The koff app values for BuGua and TFBGua were found to be 10 and times higher than that for l-Arg, and those for BuNOHG and TFBNOHG were 20 and times higher than that for NOHA Interestingly, konapp values for the fluorinated compounds TFBGua and TFBNOHG are 3.8- and 3.6-fold lower than those for their nonfluorinated analogues BuGua and BuNOHG, respectively, and the koff app values for TFBGua and TFBNOHG are 1.6 and times lower than those for BuGua and BuNOHG, respectively Discussion Binding of guanidines and N-hydroxyguanidines to iNOSoxy Fig Plots of the rates of spectral transitions observed upon the addition of guanidines or N-hydroxyguanidines to iNOSoxy in the presence of ImH vs the postmixing concentration of the studied guanidine or N-hydroxyguanidine Best linear fits are shown (A) L-Arg and NOHA, (B) N-hydroxyguanidines BuNOHG and TFBNOHG, (C) guanidines BuGua and TFBGua See Table for the structure of compounds FEBS Journal 272 (2005) 3172–3183 ª 2005 FEBS In the series of guanidines and N-hydroxyguanidines studied here, the ratio between the Kapp or Kd (calculated using Eqn 1¢) of a guanidine and that of its corresponding N-hydroxyguanidine was always found to be between 0.5 and (Table 1) This was true for pairs of compounds showing dissociation constants in the micromolar range (l-Arg ⁄ NOHA) and pairs of compounds showing Kd in the millimolar range (FPhGua ⁄ FPhNOHG) The difference between the Kapp values for the guanidines and those for the corresponding N-hydroxyguanidines was, in most cases, small and barely significant However, we found that the Kapp value for NOHA is slightly lower than that for l-Arg (Table 1), and because such an observation has also been previously reported by several authors with nNOS [12,22,23] and iNOS [10], this difference is probably significant By contrast, we found that the Km value for NOHA is higher than that for l-Arg (Table 2), also in accordance with the literature data on the three isoforms [29,31,32] In the studied series, 3177 ` D Lefevre-Groboillot et al Substrate specificity of iNOS Table Apparent association and dissociation rate constants (konapp and koff app ) for the binding of guanidines and N-hydroxyguanidines to iNOSoxy in the presence of 400 lM ImH See Table for the structure of compounds The rates of spectral transitions (Fig 2) were fitted vs the postmixing concentrations of the studied guanidine or N-hydroxyguanidine with a linear function, as shown on Fig koff app was defined as the y intercept and konapp as the slope Compounds konapp (s)1ỈM)1) koff app (s)1) L-Arg 120 000 12 000 18 000 3600 4700 1000 0.1 50 2.0 30 0.5 NOHA BuGua BuNOHG TFBGua TFBNOHG ± ± ± ± ± ± 30 000 1500 3000 200 800 200 ± ± ± ± ± ± 0.06 10 0.4 0.4 binding of an N-hydroxyguanidine moiety in the iNOS heme pocket thus roughly involves the same binding energy as binding of the guanidine moiety, and the equilibrium constants are mainly determined by the alkyl or aryl substituents of the compounds The crystal structures of mouse iNOSoxy–l-Arg and bovine eNOSoxy–l-Arg complexes [33,34] have shown that the guanidine moiety of l-Arg makes a salt bridge with the side chain of a conserved glutamate residue (E371 in mouse iNOS), and H-bonds with a backbone carbonyl oxygen atom (W366 in mouse iNOS) The crystal structures of iNOSoxy–NOHA complexes showed identical positioning of the N-hydroxyguanidine moiety of NOHA, with additional contacts between the N-hydroxyguanidine hydroxy group and the amide nitrogen of a conserved glycine (G365 in mouse iNOS) [18,19,21] The crystal structures of eNOSoxy–ClPhNOHG and nNOSoxy–BuNOHG complexes showed similar positionings of the N-hydroxyguanidine moiety of ClPhNOHG and BuNOHG involving: (a) a salt bridge between a glutamate side chain and the two nonhydroxylated nitrogens of the N-hydroxyguanidine, and (b) a nonbonded contact between the hydroxy group and a glycine nitrogen [17,19] It thus seems that in such a positioning, changing the N-hydroxyguanidine moiety into a guanidine moiety does not strongly modify the energy of binding to iNOS Preliminary data show that this is also true ` for nNOS (D Lefevre-Groboillot, unpublished data) The structure–affinity relationship for alkylguanidines and N-alkyl-N¢-hydroxyguanidines bearing nonfunctionalized linear alkyl chains (ProGua, BuGua, PentylGua, HexGua, ProNOHG, BuNOHG, PentylNOHG and HexNOHG) showed that the binding affinity is maximal for the compounds bearing a butyl chain, i.e BuNOHG and BuGua, with Kd values around 20 lm (Table 1) Compounds bearing a 3178 n-pentyl chain (PentylNOHG and PentylGua) still bind well to the iNOS active site (Kd around 100 lm) but compounds bearing an n-hexyl chain (HexNOHG and HexGua) interact with iNOS with low affinities (Kd > 150 lm) The crystal structure of the nNOSoxy– BuNOHG complex showed that the butyl chain of BuNOHG interacts with the side chain of a conserved valine residue (V567), a conserved proline (P565) and the amide moiety of a conserved glutamine (Q478) [19] Because BuNOHG has previously been reported to be similarly efficiently oxidized into NO by both iNOS and nNOS [4,6,9], the binding modes of this compound for the two isoforms is expected to be similar We measured the Kd for the binding of BuNOHG to nNOSoxy and found a somewhat higher value of 100 lm (data not shown), suggesting that the binding of BuNOHG to iNOS is favoured slightly over its binding to nNOS The Km value for the oxidation of BuNOHG by iNOS and nNOS were also found to follow the order iNOS < nNOS [4,6,9] It appears that the hydrophobic contacts such as those observed between the butyl chain and the protein in the nNOSoxy–BuNOHG crystal structure are sufficient to allow compounds BuNOHG and BuGua to bind to the active site of iNOS and nNOS with Kd values in the 20–100 lm range Interestingly, the crystal structure of the nNOSoxy–BuNOHG complex revealed that upon binding of BuNOHG the side chain of residue Q257 has to shift from its position observed in other complexes (including the nNOSoxy–NOHA complex), in order to accommodate the terminal methyl group of BuNOHG [19] This is in agreement with the fact that longer compounds such as PentylNOHG or HexNOHG showed lower affinities for the iNOS active site, because their binding may require an important reorganization of the protein environment The introduction of both an amino function and a carboxylate function on the terminal methyl group of BuGua and BuNOHG in a configuration leading to the natural substrates, l-Arg and NOHA, led to a 10-fold decrease in the observed equilibrium constants (Table 1) The positioning of the a-amino acid moiety of NOHA or l-Arg analogues appears to be critical for binding to iNOSoxy Indeed, the Kd values for l-Arg and NOHA were found to be in the 2–4 lm range, in agreement with previously reported data [10,11,13], whereas those for the longer analogues homo-l-Arg and homo-NOHA were found to be in the 10–20 lm range and that for the shorter analogue Nor-l-Arg was found to be > 300 lm This indicates that the alkyl chains of l-Arg (or NOHA) optimally position their guanidine (or N-hydroxyguanidine) and a-amino acid moieties relative to each other in the FEBS Journal 272 (2005) 3172–3183 ª 2005 FEBS ` D Lefevre-Groboillot et al NOS active site This also indicates that adding one methylene in the l-Arg chain does not impede efficient binding, whereas removal of one methylene group is detrimental for the interaction between the protein and the substrate The crystal structure of the eNOSoxy– homo-l-Arg complex (PDB entry 1DM7, C.S Raman et al 1999) actually showed that homo-l-Arg interacts with the active site of eNOSoxy in a manner similar to l-Arg, involving roughly identical positionings of the guanidine and a-amino acid moieties However, the longer alkyl chain of homo-l-Arg forms a small bulge between the two heme propionates in contact with the heme and the side chain of a conserved valine (V338) The decrease of the affinity of homo-l-Arg and homoNOHA compared with l-Arg and NOHA (Table 1) could be linked to this unfavourable bulging conformation of the alkyl chain of homo-l-Arg The simultaneous presence of both the a-amino and a-carboxylate moieties appears to be necessary because the NOHA analogue bearing only an a-amino moiety (NOHAgma) interacted with iNOSoxy with an affinity (Kd  250 lm) much lower than that found for BuNOHG, and the NOHA analogue bearing only an a-carboxylate moiety (NOHGPA) did not interact with iNOS (Kd > 500 lm) This suggests that the a-amino and a-carboxylate groups cooperate to provide favourable binding enthalpy for the formation of the complex between NOHA and the protein The crystal structures of l-Arg or NOHA in NOS active sites actually showed that the a-amino acid moiety of these compounds interacts with the protein via an H-bond network involving one or two water molecules that links the a-amino and a-carboxylate moieties one to each other, and to protein residues [18,19,21] Finally, six compounds bearing an aryl moiety, namely FPhNOHG, TolNOHG, ClPhNOHG, BzNOHG, FPhGua and ClPhGua (Table 1), exhibited Kd values > 250 lm The crystal structure of the eNOSoxy–ClPhNOHG complex showed that the phenyl ring of ClPhNOHG is in close contact with the side chain of the conserved valine, V338, and with a propionate of the heme [17] The chlorine atom is also involved in nonbonded contacts with the conserved methionine M341 Because ClPhNOHG was previously reported to be an iNOS-specific substrate [5,6], we also measured the equilibrium constants for the binding of these compounds to eNOS and nNOS (data not shown) ClPhNOHG actually displayed significantly higher affinities for the two constitutive isoforms than for iNOS: the Kd values for its binding to nNOSoxy and eNOSoxy were found to be around 50 and 95 lm, respectively, whereas that for its binding to iNOSoxy was found to be close to 350 lm Similar higher affinities FEBS Journal 272 (2005) 3172–3183 ª 2005 FEBS Substrate specificity of iNOS for nNOSoxy compared with iNOSoxy were also observed for TolNOHG, which is also an iNOS specific substrate [5,6], and for FPhNOHG, which is a substrate highly selective for iNOS [5] These results obtained with guanidines or hydroxyguanidines bearing an N-aryl moiety recall the well-documented selectivity of N-arylamidines for inhibition of nNOS vs iNOS [35] Relationship between the structure of N-hydroxyguanidines, their affinity for iNOSoxy and their oxidation by iNOS with formation of NO Previous data showed that a very large number of monosubstituted N-hydroxyguanidines R-NH-C(¼NOH)NH2 bearing an alkyl or aryl substituent R, neither too small nor too bulky, led to the detectable production of NO in the presence of iNOS [4–7,9,29] Formation of NO from the oxidation of an N-hydroxyguanidine by iNOS is thus not specific to NOHA and can occur with many N-hydroxyguanidines The rates of NO formation from the oxidation of a great number of N-alkyl- and N-aryl-N¢-hydroxyguanidines by iNOS were found to be highly dependent on their structure [4–7,9] However, the kcat values found for NO formation upon iNOS-catalysed oxidation of the seven N-hydroxyguanidines mentioned in Table varied by less than a factor 2, whereas their Kd values varied by a factor 200 (Table 1) It thus appears that the kcat of NO formation is not simply related to the affinity of the substrate for iNOS For instance, the kcat of NO formation from FPhNOHG oxidation is 83% of that found for NOHA, whereas the Kd of this substrate is 130 times higher than that of NOHA As mentioned above and shown in Table 2, very different N-hydroxyguanidines leading to similar kcat values (58–100% of that found for NOHA) showed widespread Km ⁄ Kd ratios (from  to  20) This variation may be related to that in koff, as expected by considering the relation Km ⁄ Kd ¼ + kcat ⁄ koff From a qualitative point of view, this is in agreement with the variation in koff app for NOHA (0.1 s)1), TFBNOHG (0.5 s)1) and BuNOHG (2 s)1) (Table 3), which is inversely related to that of Km ⁄ Kd for these N-hydroxyguanidines (19.3, 8.1 and 3.0 for NOHA, TFBNOHG and BuNOHG, respectively) Rigorous and quantitative correlations could not be done immediately, as Km and kcat, Kapp, konapp and koff app values were measured under different conditions for experimental reasons (different temperatures or the presence of imidazole) However, our data provide a first general basis to understand the structural factors that are 3179 ` D Lefevre-Groboillot et al Substrate specificity of iNOS necessary for guanidines and N-hydroxyguanidines to efficiently bind to iNOS Criteria for the formation of NO from the oxidation of a guanidine by iNOS Contrary to what is observed for the N-hydroxyguanidines, not all the guanidines that bind to iNOS lead to the production of NO [5–9] For example, all arylguanidines assayed to date, among them FPhGua and TolGua, have failed to lead to any detectable amount of NO, although their affinity for iNOS is not lower than that for the corresponding N-aryl-N¢-hydroxyguanidines that lead to kcat values of formation of NO as high as 83 and 69% that obtained for NOHA (Table 2) As in the case of the N-hydroxyguanidines, the kcat values of NO formation from the oxidation of guanidines not appear to be linked to the affinity of the compounds for iNOS For example, compound TFBGua led to a kcat value of NO formation of 55% that obtained with l-Arg (Table 2) even though it bound to iNOS with a Kapp value 50 times higher (Table 1) Interestingly, the kcat of production of NO by oxidation of the studied guanidines followed the same order l-Arg > homo-l-Arg  TFBGua > PentylGua > BuGua as that found for the Km ⁄ Kd ratio of the corresponding N-hydroxyguanidines: NOHA > homo-NOHA  TFBNOHG > PentylNOHG > BuNOHG (Table 2) This suggests that the variations in the kcat values found for NO formation from the guanidines could be related to those of the koff of the corresponding N-hydroxyguanidines Accordingly, the order l-Arg > TFBGua > BuGua found for the kcat of production of NO from oxidation of these guanidines corresponds well to the order NOHA > TFBNOHG > BuNOHG found for ⁄ koff app of the corresponding N-hydroxyguanidines (Table 3) These results may suggest that a key factor for a guanidine to lead to NO formation in the presence of iNOS could be the ability of the corresponding N-hydroxyguanidine to bind to the active site without being released before being further oxidized They could explain why the compound TFBGua is so far the best non a-amino acid NO precursor upon oxidation by iNOS (Table 2), because the koff app value of the corresponding N-hydroxyguanidine TFBNOHG is particularly low (Table 3) In a more general manner, our data suggest that changes in the NOS–substrate complex structure (changes of the substrate structure, but also mutation or post-translational modification of the protein) could likely lead to a shift of the activity of NOS from NO synthesis to N-hydroxyguanidine synthesis Further investigations are currently underway to test these hypotheses Our results also suggest that the 3180 rational design of guanidines as new NO donors upon in situ oxidation by NOSs should take into account both thermodynamic and kinetic characteristics of the interaction of the protein not only with the guanidine, but also with the corresponding N-hydroxyguanidine Experimental procedures Chemicals and reagents BH4 was purchased from Alexis Biochemicals (COGER, Paris, France) and l-Arg and homo-l-Arg were from Sigma (Saint-Quentin Fallavies, France) Alkylguanidines were obtained by reaction of the corresponding amine with pyrazole-1-carboxamidine hydrochloride in the presence of diisopropylethylamine following a previously described protocol [8] Arylguanidines were obtained by reaction of the amine with N,N¢-bis(tert-butyloxycarbonyl)pyrazole-1-carboxamidine followed by acidic deprotection as previously described [8] N-Hydroxyguanidines were obtained, as well as small amounts of the corresponding ureas, by the addition of hydroxylamine hydrochloride to intermediate cyanamides in anhydrous ethanol [4–7] Cyanamides were obtained from the amines by addition of BrCN in methanol containing anhydrous sodium acetate [4,5] The physicochemical characteristics of N-(n-propyl)-N¢-hydroxyguanidine, N-(n-butyl)N¢-hydroxyguanidine, N-(n-pentyl)-N¢-hydroxyguanidine, N-(n-hexyl)-N¢-hydroxyguanidine, N-(4-fluorophenyl)-N¢hydroxyguanidine, N-(4-chlorophenyl)-N¢-hydroxyguanidine, N-(4-methylphenyl)-N¢-hydroxyguanidine, N-benzyl-N¢-hydroxyguanidine, n-butylguanidine, 4,4,4-trifluorobutylguanidine and (4-methyl)phenylguanidine have been published previously [5,6,8] NOHA and homo-NOHA were synthesized as previously reported [29] Other chemicals were from Aldrich (Saint-Quentin Fallavies, France), Sigma or Across (Noisy le Grand, France) unless otherwise indicated and were of the highest purity commercially available Protein preparation iNOSoxy (amino acids 1–498) containing a six-histidine tag at its C-terminus was overexpressed in Escherichia coli and purified in the absence of BH4 as described previously [36] It was estimated to be more than 95% pure by SDS ⁄ PAGE The enzyme concentration was determined from the 444 nm absorbance of its ferrous–CO complex by using an extinction coefficient of 76 mm)1.cm)1 [37] iNOSoxy (heme concentration lm) was incubated overnight with 25 lm BH4 and mm dithiothreitol at °C before use Assessment of NO formation Initial rates of NO formation were determined at 37 °C using the classical spectrophotometric oxyhemoglobin assay FEBS Journal 272 (2005) 3172–3183 ª 2005 FEBS ` D Lefevre-Groboillot et al for NO [38] under conditions described previously [4,5,8] In some assays, the level of NO formation was measured by electron paramagnetic resonance spectroscopy following the formation of the paramagnetic ferrous mononitrosyl diethyldithiocarbamate complex under previously described conditions [8,39] Determination of the dissociation constants for the complexes between BH4-containing iNOSoxy and guanidines or N-hydroxyguanidines Studies were carried out at room temperature in an UVIKON 942 spectrophotometer (Kontron Biotek), in a 1-cm path length cuvettes (150 lL total volume) Each cuvette contained lm iNOSoxy in 50 mm Hepes buffer, pH 7.4, in the presence of 25 lm BH4 and mm dithiothreitol The amplitude of the observed difference spectra DA(kmax ) kmin) induced by the progressive addition of ImH to an iNOSoxy solution was fitted (a) vs [ImH] with a hyperbolic function, and (b) ⁄ DA(kmax ) kmin) vs ⁄ [ImH] with a linear function The two fits gave dissociation constants (KImH) for the NOS–ImH complex that were less different than the values obtained from two identical experiments, indicating that a single binding site model satisfactorily accounted for the binding of ImH to iNOSoxy A dissociation constant value of KImH ¼ 52 ± lm was found, very close to the values reported by others [11–13] Maximum amplitude of the difference spectrum was 68 ± mm)1 cm)1 The study of the binding of guanidines and N-hydroxyguanidines to lm iNOSoxy was performed in the presence of 400 lm ImH, a situation that allows the monitoring by UV ⁄ Vis difference spectroscopy of the formation of a complex between the protein and compounds which bind to the substrate binding site [11,12,14,22,25,36,40–42] The studied guanidine or N-hydroxyguanidine (dissolved in buffer) was added stepwise to the sample cuvette, and equivalent volumes of buffer were added to the reference cuvette All experiments were carried out under conditions where the concentration of bound ligand was much smaller than the total concentration of ligand In a first calculation, the amplitude of the observed difference spectra DA(kmax ) kmin) was fitted vs the guanidine or N-hydroxyguanidine concentration [ligand] with a hyperbolic function In a second step, ⁄ DA(kmax ) kmin) was fitted vs ⁄ [ligand] with a linear function The two fits always gave apparent equilibrium constants (Kapp) for the NOS–ligand complexes that were less different than the values obtained from two identical experiments This indicated that a single binding site model satisfactorily accounted for the observed spectral changes The maximum amplitude of the difference spectra was 56 ± mm)1 cm)1 and did not vary significantly with the structure of the guanidine or N-hydroxyguanidine FEBS Journal 272 (2005) 3172–3183 ª 2005 FEBS Substrate specificity of iNOS Rapid kinetic studies of the binding of guanidines and N-hydroxyguanidines to iNOSoxy Experiments were performed at 15 °C using a stopped-flow instrument equipped with a rapid-scanning diode array detector (Hi-Tech MG 6000) and following a protocol previously described [23] A solution of iNOSoxy containing 400 lm ImH was mixed with a solution of guanidine (or N-hydroxyguanidine) also containing 400 lm ImH Postmixing heme concentration was lm The reaction was monitored by following the absorbance at 430 and 392 nm Variations of the absorbances at these two wavelengths were fitted with monoexponential functions Observed rate constants kobs were obtained by averaging the values of the rate constants measured at the two wavelengths over 5–10 shots Acknowledgements The authors thank Sylvie Dijols (UMR 8601 CNRS, Paris) for the synthesis of the guanidines and N-hydroxyguanidines used in this study DL-G thanks Zhi-Qiang Wang, Chin-Chuan Wei and Koustubh Panda (Cleveland Clinic Foundation) for their help with the stopped-flow experiments, and Jerome Santolini ˆ (CEA Saclay, France) for his help in the preparation of proteins and helpful discussions This work was supported by the French Ministry of Research (fellowship grant to DL-G), and by National Institutes of Health (grant CA53914 to DJS) References Stuehr DJ (1999) Mammalian nitric oxide synthases Biochim Biophys Acta 1411, 217–230 Pfeiffer S, Mayer B & Hemmens B (1999) Nitric oxide: chemical puzzles posed by a biological messenger Angew Chem Int Ed 38, 1714–1731 Alderton WK, Cooper CE & Knowles RG (2001) Nitric oxide synthases: structure, function and inhibition Biochem J 357, 593–615 Dijols S, Perollier C, Lefevre-Groboillot D, Pethe S, Attias R, Boucher J-L, Stuehr DJ & Mansuy D (2001) Oxidation of Nx-hydroxyarginine analogues by NO-synthase: the simple, non amino acid N-butyl-N’-hydroxyguanidine is almost as efficient an NO precursor as 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bidomain structure and successful reconstitution of catalytic activity from two separate domains generated by a baculovirus expression system J Biol Chem 271, 14631– 14635 Rodriguez-Crespo I, Gerber NC & Ortiz de Montellano PR (1996) Endothelial nitric-oxide synthase Expression in Escherichia coli, spectroscopic characterization, and role of tetrahydrobiopterin in dimer formation J Biol Chem 271, 11462–11467 3183 ... related to the binding of l-Arg [10–14] and NOHA [10], nothing is known about the thermodynamics and kinetics of the binding of guanidines and N-hydroxyguanidines to iNOS Removal of the a-amino or... from the conversion of the LS NOS–Fe(III)–ImH complex to the HS NOS–Fe(III)–l-Arg complex was observed Inhibition of the binding of l-Arg to iNOSoxy by ImH, and of the iNOS-catalysed conversion of. .. BuNOHG to iNOS is favoured slightly over its binding to nNOS The Km value for the oxidation of BuNOHG by iNOS and nNOS were also found to follow the order iNOS < nNOS [4,6,9] It appears that the