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Interactions of imidazoline ligands with the active site of purified monoamine oxidase A Tadeusz Z. E. Jones 1 , Laura Giurato 2 , Salvatore Guccione 2 and Rona R. Ramsay 1 1 Centre for Biomolecular Sciences, University of St Andrews, North Haugh, St Andrews, UK 2 Dipartimento di Scienze Farmaceutiche, Facolta ` di Farmacia, Universita ` degli Studi di Catania, Ed. 2 Citta ` Universitaria, Catania, Italy In 1984, Bousquet et al. [1] found that clonidine pro- duced a pharmacologic response, independent of a-adrenoceptors, at high-affinity imidazoline-binding sites. These binding sites were later classified into three different subtypes, and some progress was made in determining their identity and function [2,3]. Ligands to the type 2 binding sites (I 2 BS) [4] also inhibit monoamine oxidase (MAO) but with lower affinity [5]. Some MAO substrates and inhibitors bind to I 2 BS [6]. Harmane, a b-carboline formed from tryptamine, has nanomolar affinity both for I 2 BS and for MAO, and mimics the hypotensive effect of clonidine [7]. How- ever, a careful study using b-carboline derivatives showed no correlation between MAO-A inhibition and binding to either high- or low-affinity I 2 BS [8]. Both MAO and I 2 BS are located on the outer mit- ochondrial membrane [9] and show coexpression dur- ing the aging process in humans [10]. The protein labeled by I 2 BS ligands has a similar molecular weight and peptide sequence to MAO [6,11]. Transfection of yeast cells with MAO led to the expression of imidazo- line-binding sites not previously observed, although the correlation between the number of binding sites and MAO activity was poor [6,12]. However, a decrease in the number of I 2 BS in suicide victims was not accom- panied by a decrease in MAO-B [13]. Furthermore, MAO-A knockout mice did not lose the high-affinity imidazoline binding, although a peptide ( 60 kDa) was no longer labeled by the covalent imidazoline ligand [14]. At the protein level, photoaffinity labeling Keywords docking; I 2 binding sites; imidazoline; kinetics; monoamine oxidase Correspondence R. R. Ramsay, Centre for Biomolecular Sciences, University of St Andrews, North Haugh, St Andrews, Fife KY16 9ST, UK Fax: +44 1334 463400 Tel: +44 1334 463411 E-mail: rrr@st-and.ac.uk (Received 26 September 2006, revised 15 January 2007, accepted 17 January 2007) doi:10.1111/j.1742-4658.2007.05704.x The two forms of monoamine oxidase, monoamine oxidase A and mono- amine oxidase B, have been associated with imidazoline-binding sites (type 2). Imidazoline ligands saturate the imidazoline-binding sites at nanomolar concentrations, but inhibit monoamine oxidase activity only at micromolar concentrations, suggesting two different binding sites [Ozaita A, Olmos G, Boronat MA, Lizcano JM, Unzeta M & Garcı ´ a-Sevilla JA (1997) Br J Pharmacol 121, 901–912]. When purified human monoamine oxidase A was used to examine the interaction with the active site, inhibition by guana- benz, 2-(2-benzofuranyl)-2-imidazoline and idazoxan was competitive with kynuramine as substrate, giving K i values of 3 lm,26lm and 125 lm, respectively. Titration of monoamine oxidase A with imidazoline ligands induced spectral changes that were used to measure the binding affinities for guanabenz (19.3 ± 3.9 lm) and 2-(2-benzofuranyl)-2-imidazoline (49 ± 8 lm). Only one type of binding site was detected. Agmatine, a putative endogenous ligand for some imidazoline sites, reduced monoamine oxidase A under anaerobic conditions, indicating that it binds close to the flavin in the active site. Flexible docking studies revealed multiple orienta- tions within the large active site, including orientations close to the flavin that would allow oxidation of agmatine. Abbreviations 2-BFI, 2-(2-benzofuranyl)-2-imidazoline; I 2 BS, imidazoline-binding site (type 2); MAO, monoamine oxidase; MPTP, 1-methyl-4-phenyl-1,2,3,6- tetrahydropyridine. FEBS Journal 274 (2007) 1567–1575 ª 2007 The Authors Journal compilation ª 2007 FEBS 1567 of the imidazoline site on MAO-B identified a labeled peptide containing amino acids 149–222 of the MAO-B sequence [12]. The crystal structure of MAO-B shows that some of the residues in this peptide lie between the entrance cavity and the active site [15]. On the other hand, inhibition of MAO with a mechanism- based irreversible inhibitor did not prevent binding of the I 2 ligand 2-(2-benzofuranyl)-2-imidazoline (2-BFI), suggesting that the I 2 BS was not the active site of MAO [16]. Imidazolines reversibly inhibit MAO, but with IC 50 or K i values in the micromolar range [5,6,17–20]. Examples of the literature values for MAO inhibition measured in membranous samples with the ligands used in this study are included in Table 1. It has been proposed that I 2 BS ligands bind both to the MAO act- ive site, thereby inhibiting the enzyme, and to a second site with substantially higher affinity present on a sub- population of MAO-B enzymes [21]. Recently, some imidazole derivatives were reported to have high I 2 BS affinity but negligible MAO inhibitory activity [22], reinforcing a separate identity of the I 2 BS from the MAO active site. The kinetics of MAO inhibition by imidazolines have been described as noncompetitive [6,20], noncom- petitive and mixed [17], or competitive for MAO-A inhibition and mixed for MAO-B inhibition [5]. This study examines binding of the imidazoline ligands to purified enzyme of a single subtype (MAO-A) to characterize their active site binding and the resulting inhibition. The use of purified enzyme simplifies the system for kinetics and also allows observation of the spectrum of the flavin cofactor in MAO-A. The subtle changes in the flavin spectrum on ligand binding in the active site [23,24] are concentration-dependent and saturable, permitting calculation of the dissociation constant for binding to the active site of MAO-A. To complement these two methods, flexible docking [25] was used to visualize the interactions between imidazo- lines and the MAO-A active site. Results Kinetics of inhibition Guanabenz, 2-BFI and idazoxan all act as inhibitors of MAO-A, but no inhibition was seen below 0.1 lm (Fig. 1). Reported K D values for I 2 BS vary consider- ably [5,6,18,19,26], but all indicate much more than 50% occupancy of the I 2 BS at 0.1 lm. There was also no activation of MAO-A by any ligand, either with kynuramine (Fig. 1) or with 1-methyl-4-phenyl-1,2,3,6- tetrahydropyridine (MPTP) (not shown) as a sub- strate, in contrast to the activation by imidazolines of benzylamine oxidation catalyzed by bovine plasma semicarbazide-sensitive amine oxidase [27]. Allosteric activation of semicarbazide-sensitive amine oxidase by imidazolines was seen only with selected substrates, so both kynuramine and MPTP were tested for MAO-A. As some MAO-A inhibitors (such as the b-carbolines [28], also known to bind to both I 2 BS and MAO-A [8]) are time-dependent, the time dependence of inhibition was investigated. For 2-BFI, the inhibition immediately upon mixing was 40 ± 3% (n ¼ 3). When 2-BFI was incubated with the enzyme in the cuvette before addi- tion of substrate, the inhibition was 37 ± 5% (n ¼ 4) after either 1 min or 5 min of preincubation. A similar lack of any time-dependent increase in inhibition was found for the other two inhibitors. Thus, there is no tighter, time-dependent binding that might account for the discrepancy between the reported affinities for the I 2 BS and the MAO-A active site. Table 1. Binding and inhibition constants for I 2 ligands with MAO-A. The K i values for purified human MAO-A were determined as des- cribed in Experimental procedures. The K D values for human MAO-A were calculated from the absorbance change at 500 nm induced by ligand binding (from two experiments for guanabenz and one for 2-BFI and idazoxan, each with 14–18 points). The literature values for inhibition of MAO-A were measured in membranes from rat liver. ND, not determined. Ligand This work Literature values K i (lM) K i (lM) K D (lM) IC 50 (lM) [5] IC 50 (lM) [9] MPTP Kynuramine - Serotonin Serotonin Guanabenz 7 ± 2 3 ± 2 19.3 ± 3.9 4 ND 2-BFI 40 ± 8 26 ± 3 49 ± 8 11 16.5 Idazoxan 165 ± 30 125 ± 40 > 100 280 220 Guanabenz Idazoxan % Inhibition –9 100 80 60 40 20 0 –8 –7 –6 Log ([I],M) –5 –4 –3 2-BFI Fig. 1. Inhibition of MAO-A by guanabenz (solid line), 2-BFI (dotted line), and idazoxan (dashed line). The activity of MAO-A was deter- mined with 0.5 m M kynuramine (4 · K m ). The unit for the inhibitor concentration is molÆL )1 . Imidazoline binding in the MAO-A active site T. Z. E. Jones et al. 1568 FEBS Journal 274 (2007) 1567–1575 ª 2007 The Authors Journal compilation ª 2007 FEBS When MAO-A was assayed with kynuramine at 0.5 mm (4 · K m ), the IC 50 values obtained were 6 lm for guanabenz, 48 lm for 2-BFI, and over 500 lm for idazoxan, giving a good range of affinity suitable for the binding studies. These values are consistent with the IC 50 values in the literature for membrane-bound enzyme (Table 1). The type of inhibition and the inhi- bitory constants (K i ) were determined by varying both substrate and inhibitor (see Experimental procedures). The Lineweaver–Burk plot used for illustration in Fig. 2 shows that the inhibition was competitive with the amine substrate. The K i values are given in Table 1. There are only small differences between the values obtained with the two substrates, despite the different steady-state level of oxidized enzyme available for inhibitor binding with kynuramine and MPTP [29]. Difference spectra and binding curves When ligands bind to the active site of MAO-A, the environment of the flavin is altered, resulting in spec- tral changes [24]. The I 2 BS ligands caused similar, sat- urable changes in the MAO-A spectrum (shown for guanabenz in Fig. 3), suggesting that these I 2 BS lig- ands might be expected to bind in the active site in the same way as other reversible MAO-A inhibitors. In the experiment in Fig. 3, the extinction coefficient for guanabenz from the change at 500 nm was 1913 m )1 Æcm )1 , and the dissociation constant calcula- ted directly from the change in absorbance of MAO-A after addition of guanabenz fitted to a rectangular hyperbola was 20.3 ± 1.3 lm. The mean K D value for guanabenz and the values for 2-BFI and idazoxan are given in Table 1. Evidence for the proximity of these ligands to the flavin in MAO-A comes from the influence of the lig- and on the redox properties of the flavin. Like other inhibitors of MAO-A [23,30], 2-BFI strongly increased the amount of anionic flavosemiquinone observed at 412 nm during reduction of the flavin in MAO-A by dithionite (Fig. 4A). In contrast, agmatine [(4-amino- butyl)guanidine], the endogenous compound that binds to some imidazoline sites [31,32], is a substrate for MAO-A. Figure 4B shows the reduction of anaerobic MAO-A by agmatine without addition of chemical Fig. 2. Inhibition of MAO-A by guanabenz is competitive. Kynuram- ine was varied (0.1–0.9 m M) to determine the apparent K m in the absence (solid squares) and presence (other symbols) of inhibitor at the concentrations indicated. Secondary plots of the apparent K m against the inhibitor concentration were used to determine the K i values given in Table 1. The inhibition is shown as Lineweaver– Burk plots, prepared by plotting the reciprocal data in CRICKETGRAPH, to illustrate the unchanged V max . 0.02A B 0.02 –0.02 –0.03 0.03 300 400 Wavelength (nm) Absorbance change Absorbance change 500 600 0.01 0.01 –0.01 0 0 02040 [Guanabenz] µ M 60 80 Fig. 3. Spectral changes on ligand binding to MAO-A. (A) Selected difference spectra (the spectrum for MAO-A alone subtracted from the spectrum for MAO-A + inhibitor) are shown for guanabenz (17 l M MAO-A with 8, 16, 32 and 72 lM guanabenz). (B) The satu- rable changes at 500 nm indicate the amount of enzyme with ligand bound. The line fitted to the data is a rectangular hyperbola. T. Z. E. Jones et al. Imidazoline binding in the MAO-A active site FEBS Journal 274 (2007) 1567–1575 ª 2007 The Authors Journal compilation ª 2007 FEBS 1569 reductant. As with all substrates, no semiquinone is observed. The transfer of electrons from the amine to the flavin requires a catalytic interaction, so the nitro- genous group of agmatine must be within 3 A ˚ of N5 of the isoalloxazine ring of the flavin. Agmatine, however, binds poorly to MAO-A, giving only 50% inhibition of the oxidation of 0.3 mm kynuramine at 1 mm. Docking of imidazolines to the structure of the MAO-A active site Using qxp ⁄ flo software [33], three imidazoline com- pounds were docked into the human MAO-A structure (Protein Data Bank code 2BXS, 3.15 A ˚ [25]). The cov- alently bound clorgyline present in the structure of MAO-A was removed. Hydrogens were added to the FAD to reflect the oxidized state of unligated enzyme. Residues lining the site (listed in Experimental proce- dures) were allowed to move to give flexibility to the binding site. The initial energy-minimized orientations for guanabenz, 2-BFI and idazoxan placed the com- pounds at the entrance of the active site cavity near amino acid residues found in the peptide photolabeled with an I 2 BS ligand. This would be sufficient to block access of substrate and so inhibit the enzyme. How- ever, this position is too far from the flavin for the reaction that takes place with agmatine. Alternative, higher-energy orientations placed the ligands near the flavin in MAO-A, similar to the position previously found for linezolid, an oxazolidinone inhibitor of MAO-A [24], and as expected for ligands that alter the spectrum of the enzyme. In order to compare the conformation of all three ligands near the flavin in MAO-A, the docking was repeated with the compounds bound through a zero- order bond to C4a of the flavin molecule. The results obtained under this condition are shown in Fig. 5. The imidazoline groups of 2-BFI and idazoxan may form a hydrophobic interaction with Tyr407, which is about 3.30 A ˚ away. The flexibility of guanabenz allowed sev- eral orientations equally close to the flavin in two binding modes. In one mode, its aromatic ring occu- pies the same area as the aromatic rings of 2-BFI and idazoxan (Fig. 5A,B) in the MAO-A active site area outlined by residues Tyr69, Tyr197, Phe208, Tyr407, Phe352, Tyr444, and the isoalloxazine ring. Guanabenz also assumes an alternative binding mode with respect to the other ligands (Fig. 5C), with the guanidinium group bent away from the tyrosines. This alternative binding mode may indicate an enhanced probability of Fig. 4. Inhibitor and substrate imidazolines have opposite effects on stabilization of the anionic semiquinone. (A) During reduction of MAO-A (17 l M) by dithionite, the presence of 2-BFI (2.5 mM) increases the yield of red anionic semiquinone at 414 nm. (B) Agm- atine reduces MAO-A (18 l M) without the addition of dithionite, but no semiquinone peak at 414 nm is seen. Additions are 0.12, 0.16, 0.20, 0.40, 0.50 and 1.0 m M agmatine over 4 h. B FAD CYS 406 TYR 69 TYR 407 GLU 216 TYR 444 TYR 197 ASN 181 PHE 352 PHE 208 C FAD CYS 406 TYR 69 TYR 407 GLU 216 TYR 444 TYR 197 ASN 181 PHE 352 PHE 208 A CYS 406 TYR 197 TYR 444 TYR 407 FAD TYR 69 PHE 352 PHE 208 GLU 216 ASN 181 Fig. 5. Binding of the stereoisomers of 2-BFI (A) and idazoxan (B), and of guanabenz (C) to MAO-A. The active site of MAO-A (2BXS) was prepared as described in Experimental procedures. Residues allowed flexibility are shown in purple, and the flavin is orange. The R forms of the ligands are green and the S forms are yellow. For 2-BFI, the oxygen atoms in the heterocycle are marked by a circle. Imidazoline binding in the MAO-A active site T. Z. E. Jones et al. 1570 FEBS Journal 274 (2007) 1567–1575 ª 2007 The Authors Journal compilation ª 2007 FEBS good interactions with the enzyme, and so might explain the better K i value of guanabenz (Table 1). Interestingly, the result of docking a positively charged (protonated) guanabenz was the same as for the un- charged molecule. At the pH of the assay and physio- logically, most of the guanabenz would be in the protonated, positively charged form. Docking of 2-BFI and idazoxan was performed for both enantiomers. The orientations for the R (green) and S (yellow) forms of 2-BFI and idazoxan in Fig. 5A,B show relatively similar localization for the imidazole ring, with the rest of the molecule easily accommodated in the relatively large cavity of MAO-A. Figure 6 shows that the optimum position of neutral agmatine in MAO-A is consistent with the reduction observed experimentally, as the primary amino group is between the two tyrosines, with the amino nitrogen almost close enough to act as a substrate (mean dis- tance in the three best orientations was 4 A ˚ ). All the three possible forms of agmatine were docked into MAO-A: neutral, protonated, and diprotonated. Although the three forms will be in equilibrium, the pK a value of 8.93 for the amino group [34] means that the diprotonated form will be the most abundant at the assay pH of 7.2. With a pK a of 12.48, the guanidinium group is positively charged (protonated) in most envi- ronments but, because of the conjugation between the double bond and the nitrogen lone pairs, the positive charge is delocalized on the three nitrogens [35]. On the basis of titrations in octanol, it has been suggested that compounds such as guanidine retain basicity in low- dielectric medium to a greater extent than does a simple amine [36]. The guanidinium group is also able to form multiple hydrogen bonds [34]. The conformational searches, carried out with macromodel [37], highlighted the fact that the diprotonated form is the most stable () 1325 kJÆmol )1 ), followed by the mono- protonated form () 1070 kJÆmol )1 ) and by the neutral form () 705 kJÆmol )1 ). Despite the different charges, the free energies derived from the docking results were very close one another, with a maximum difference of about 6 kJÆmol )1 between the neutral form (best) and the diprotonated one. The slightly better value for the neutral form could be explained by analysis of the ener- getic contributions: the negative hydrophobic term is lower for the more lipophilic neutral structure, and for the protonated forms the positive term for the polar desolvation is higher, making the total interaction energy less favorable. This insight is useful because the poor inhibition of MAO-A by agmatine (IC 50 of 1mm at 0.3 mm kynuramine) makes further studies impractical. To investigate whether the guanidinium or amino group would approach closer to the flavin in a less flexible molecule, the serum protease inhibitor 4-amino- benzamidine was docked to MAO-A. The same com- ments as were made for agmatine apply to the charges on this structure. As with agmatine, either orientation was possible. When the amino group was deprotonated in either agmatine or 4-aminobenzamidine, it was more likely to lie between the two tyrosines close to the flavin than the guanidinium group, but again, energy differ- ences were small. Discussion The association of MAO with I 2 BS was established by the appearance of an I 2 BS in yeast expressing human MAO-A or MAO-B [6], but high-affinity, specific bind- ing of [ 3 H]idazoxan was not altered in MAO-A knock- out mice, although covalent labeling of one peptide was lost [14]. The irreversible inhibition of MOA in vivo has been shown to reduce the density of imida- zoline-binding sites in rat brain [38,39]. Here we have used kinetic and spectral techniques to characterize the binding of imidazolines to the active site of purified MAO-A. The representative I 2 BS ligands used are lin- ear competitive inhibitors of purified MAO-A, in agreement with the data of Ozaita et al. (1997) for membrane-bound MAO-A and MAO-B [5]. Spectral changes and the alteration of the redox properties of the flavin (Figs 2 and 3) confirm that I 2 BS ligands bind FAD CYS 406 PHE 352 PHE 208 TYR 69 TYR 407 TYR 444 ASN 181 TYR 197 GLU 216 Fig. 6. Agmatine in the active site of MAO-A. Binding of the neut- ral form of agmatine (green) close to the flavin in MAO-A is stabil- ized by the hydrogen bonds with Glu216, Tyr444, Tyr197, and Asn181. Colours are as in Fig. 5. T. Z. E. Jones et al. Imidazoline binding in the MAO-A active site FEBS Journal 274 (2007) 1567–1575 ª 2007 The Authors Journal compilation ª 2007 FEBS 1571 at the active site. Typical changes in the flavin spec- trum are seen on binding of I 2 BS ligands, and the K D values calculated from the saturable decreases in absorbance at 500 nm were slightly higher than the kinetic K i values (Table 1). The spectral change requires close association of the ligand with the flavin, whereas competitive inhibition requires only blocking of access to the active site. The multiple orientations seen for guanabenz in the docking study included some that are less likely to influence the flavin and the adja- cent tyrosine rings directly in the way seen for 2-BFI. This may be an explanation for the higher K D than K i with guanabenz. The reduction of MAO-A by agmatine (Fig. 4B) establishes that the ligand can bind near the flavin, because proximity is required for electron transfer. However, the very poor inhibition of kynuramine oxi- dation by agmatine (50% inhibition at 1 mm in the presence of 0.3 mm kynuramine) and its very slow reduction of anaerobic MAO-A indicate that MAO-A is not a pathway for agmatine metabolism. The copper- containing amine oxidases are more likely to contribute to the oxidation of this biologically active amine [35]. Inhibition of MAO-A by I 2 BS ligands is shown here to require over 1000-fold higher concentrations than that reported in the literature for saturation of I 2 BS [6]. Binding to the active site of solubilized MAO-A has been measured directly (Fig. 3); this revealed only one site with the same low affinity as found for both MAO-A and MAO-B in membranous preparations [5]. These differences add to the evidence that the I 2 BS is distinct from the active site of MAO. The flexible docking models (Fig. 5) revealed that all three I 2 BS ligands were easily accommodated in the hydrophobic region surrounded by residues Tyr69, Tyr197, Phe208, Tyr407, Phe352, Tyr444, and the iso- alloxazine ring, with rather small differences in free energy that did not reflect the differences in K D values. The optimal orientation for guanabenz in MAO-A has the closest nitrogen atom less than 5 A ˚ from N5 of the isoalloxazine ring. This orientation and the alternative binding modes found may explain why it is the best of the inhibitors examined here. Given that all three ligands tested had similar ener- gies despite their well-separated K i values, no conclu- sions can be drawn from the similar energies and orientations for the R and S stereoisomers of both 2-BFI and idazoxan. As only the racemic mixture was available, discrimination of the stereoisomers by MAO-A was not tested experimentally. The I 2 BS does show discrimination between the (+) and (–) forms of idazoxan, with the (–) isomer more potent on periph- eral I 2 sites and the (+) isomer more potent on central I 2 BS sites [40,41]. Further experimental data on both MAO and the I 2 BS stereospecificities would be useful. Interestingly, the lowest-energy orientation for each of the three ligands in the large active site of MAO-A put them in the middle of the active site pocket, adja- cent to residues in the peptide identified by photoaffin- ity labeling [12]. This suggests that the conclusion that this peptide forms part of the I 2 BS location rather than just the active site must be taken with caution. In conclusion, this study has characterized the bind- ing to MAO-A of three commonly used I 2 BS ligands, and had provided evidence for only one binding site adjacent to the flavin in this isolated preparation. All three show three orders of magnitude lower affinity for the active site than reported values for I 2 BS. It seems clear that the active site of MAO is not the site to which I 2 BS ligands bind with nanomolar affinity. However, imidazolines do bind at the active site of MAO-A with dissociation constants in the 10–200 lm range, causing spectral and redox changes as seen for other active site inhibitors. The modeling studies revealed multiple ori- entations within the hydrophobic active site that did not differ much with the protonation state and provi- ded a possible explanation for the covalent labeling of an active site peptide in MAO by an imidazoline ligand. Experimental procedures Materials MOA-A (human liver form) expressed in Saccharomyces cerevisiae [42] was purified and stored at ) 20 °C in a solu- tion of 50 mm potassium phosphate (pH 7.2), 0.8% n-octyl- b-d-glucopyranoside (Melford Laboratories Ltd, Ipswich, UK), 1.5 m m dithiothreitol, 0.5 mmd-amphetamine, and 50% glycerol. The specific activity was 1 lmolÆmin )1 Æ(mg protein) )1 with kynuramine as the substrate. Only one major peak was seen in the mass spectrum, and the purity was greater than 95% as determined by silver-stained SDS gel electrophoresis. Before use, dithiothreitol, d-amphetam- ine and glycerol were removed by gel filtration in a spin col- umn of G-50 Sephadex equilibrated with 50 mm potassium phosphate (pH 7.2) containing detergent (0.05% Brij) [30]. 2-BFI was purchased from Tocris (Bristol, UK), and all other chemicals were obtained from Sigma-Aldrich Co. Ltd. (Poole, UK). MAO-A assays and spectral titrations Initial rates of oxidation were measured spectrophotometri- cally at 30 °Cin50mm potassium phosphate (pH 7.2) con- taining 0.05% Triton X-100. K i values were determined from three to five separate experiments, using a substrate Imidazoline binding in the MAO-A active site T. Z. E. Jones et al. 1572 FEBS Journal 274 (2007) 1567–1575 ª 2007 The Authors Journal compilation ª 2007 FEBS range of 0.1–0.9 mm kynuramine or 0.025–0.5 mm MPTP at six different inhibitor concentrations over a 10-fold range. The formation of the product was followed spectro- photometrically at 314 nm in assays with kynuramine [43] and at 343 nm with MPTP [44]. The kinetic constants were obtained by direct fit for each group of six substrate con- centrations in duplicate in the Shimadzu kinetics program of the UV-2101PC spectrophotometer (Shimadzu UK Ltd., Milton Keynes, UK). The linear secondary plots were fitted in Excel. Spectra were recorded approximately 5 min after each addition in a Shimadzu UV-2101PC spectrophotometer in an anaerobic cuvette containing MAO-A at about 10 lm in 50 mm potassium phosphate (pH 7.2) containing 0.05% Brij. The change in absorbance at 500 nm was used to determine the binding parameters, fitting the data to a rect- angular hyperbola in enzfitter. Modeling The molecular modeling studies were carried on an SGI- OCTANE R12000 workstation operating under irix 6.5+ (Silicon Graphics Inc., Sunnyvale, CA, USA). The ligand structures were built through the building tool of the soft- ware macromodel version 8.1 [37] (Schrodinger, Inc., Port- land OR, USA; http://www.schrodinger.com). Aqueous conformational analyses by macromodel were performed using the Monte Carlo Multiple Minimum (MCMM) Search protocol with the Merck Molecular Force Field (MMFFs). Prior to submitting the ligands to the search protocol, a minimization was carried out using the MMFFs as implemented in macromodel. Default options were used with the Polak–Ribiere Conjugate Gradient scheme, until a gradient of 0.001 kJÆA ˚ )1 mol was reached. To search the conformational space, 5000 Monte Carlo steps were per- formed on each starting conformation. An energy cut-off of 20.0 kJÆmol )1 ,highenoughtomaptheconformational space, including the bioactive conformation, was applied to the search results. The lowest-energy conformer of each ligand was exported to Protein Data Bank format to be read by the docking software. Docking studies were performed using the qxp ⁄ flo soft- ware using the qxp mcdock+ module (1000 cycles). qxp is the molecular mechanics module in flo+ (version April03), a molecular design program (Thistlesoft, Cole- brook, CT, USA) [33]. The enzyme structure used for the calculations was the human MAO-A (Protein Data Bank code 2BXS, 3.15 A ˚ [25]). The covalently bound clorgyline present in the struc- ture of MAO-A was removed. Hydrogens were added to the FAD to reflect the oxidized state of unligated enzyme. To give flexibility to the binding site, residues lining the site were allowed to move. These residues were: Cys406 (to which the flavin is covalently bound), Tyr69, Tyr444, Tyr407, Phe208, Phe352, Tyr197, Asn181, and Glu216. To speed the calculations, the enzyme was cut to 12 A ˚ around the active site before each docking. The docking procedure was tested using clorgyline as a control for comparison with the original structure. When the covalent bond was present, the optimum position was in the same area as in the crystal structure. With oxidized FAD and a zero-order bond to the flavin, binding was similar but not exactly superimposable. In the absence of any bond, the clorgyline molecule moved away from the flavin. Acknowledgements T. Z. E. Jones and R. R. Ramsay thank AstraZeneca for support. Dr Colin McMartin (Thistlesoft, Cole- brook, CT, USA) is gratefully acknowledged for the software flo+ (version April03). We thank Dr Robert A. Scherrer (BIOpK, White Bear Lake, MN, USA) for helpful discussion and suggestions on the physicochem- ical properties of the guanidine derivatives. The mode- ling work is part of Laura Giurato’s PhD thesis, to be presented at the Universita ` degli Studi di Catania. References 1 Bousquet P, Feldman J & Schwartz J (1984) Central cardiovascular effects of alpha-adrenergic drugs ) dif- ferences between catecholamines and imidazolines. 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