Báo cáo khoa học: An important lysine residue in copper⁄quinone-containing amine oxidases doc

An important lysine residue in copper ⁄ quinone-containing amine oxidases Anna Mura 1 , Roberto Anedda 2 , Francesca Pintus 1 , Mariano Casu 2 , Alessandra Padiglia 1 , Giovanni Floris 1 and Rosaria Medda 1 1 Department of Applied Sciences in Biosystems, University of Cagliari, Italy 2 Department of Chemical Science, University of Cagliari, Italy Copper ⁄ quinone-containing amine oxidases [(deami- nating) (copper-containing) amine:oxygen oxidoreduc- tase; EC 1.4.3.6] (Cu ⁄ TPQ AOs) found in bacteria, yeasts, fungi, plants and mammals catalyze the oxida- tive deamination of primary amines to the correspond- ing aldehydes while reducing molecular oxygen to hydrogen peroxide [1]. The ping-pong catalytic mech- anism of Cu ⁄ TPQ AOs can basically be divided into two half-reactions. One, referred to as a ‘reductive half-reaction’, involves the oxidation of amine to aldehyde and the formation of a reduced form of the TPQ cofactor: E ox þ R À CH 2 À NH þ 3 ! E red þ R À CHO The other, known as ‘the oxidative half-reaction’, involves the reoxidation of the enzyme and contem- poraneous release of ammonia and hydrogen peroxide: Keywords amine oxidase; copper; NMR; quinoprotein; xenon Correspondence R. Medda, Department of Applied Sciences in Biosystems, University of Cagliari, Cittadella Universitaria, I-09042 Monserrato (CA), Italy Fax: +39 070 6754524 Tel: +39 070 6754517 E-mail: rmedda@unica.it (Received 4 October 2006, revised 27 February 2007, accepted 15 March 2007) doi:10.1111/j.1742-4658.2007.05793.x The interaction of xenon with copper ⁄ 6-hydroxydopa (2,4,5-trihydroxy- phenethylamine) quinone (TPQ) amine oxidases from the plant pulses lentil (Lens esculenta) and pea (Pisum sativum) (seedlings), the perennial Mediter- ranean shrub Euphorbia characias (latex), and the mammals cattle (serum) and pigs (kidney), were investigated by NMR and optical spectroscopy of the aqueous solutions of the enzymes. 129 Xe chemical shift provided evi- dence of xenon binding to one or more cavities of all these enzymes, and optical spectroscopy showed that under 10 atm of xenon gas, and in the absence of a substrate, the plant enzyme cofactor (TPQ), is converted into its reduced semiquinolamine radical. The kinetic parameters of the ana- lyzed plant amine oxidases showed that the k c value of the xenon-treated enzymes was reduced by 40%. Moreover, whereas the measured K m value for oxygen and for the aromatic monoamine benzylamine was shown to be unchanged, the K m value for the diamine putrescine increased remarkably after the addition of xenon. Under the same experimental conditions, the TPQ of bovine serum amine oxidase maintained its oxidized form, whereas in pig kidney, the reduced aminoquinol species was formed without the radical species. Moreover the k c value of the xenon-treated pig enzyme in the presence of both benzylamine and cadaverine was shown to be dramat- ically reduced. It is proposed that the lysine residue at the active site of amine oxidase could be involved both in the formation of the reduced TPQ and in controlling catalytic activity. Abbreviations AO, amine oxidase; AGAO, Arthrobacter globiformis amine oxidase; BSAO, bovine serum amine oxidase; Cu-AO, copper amine oxidase; DABY, 1,4-diamino-2-butyne; ELAO, Euphorbia characias amine oxidase; HPAO, Hansenula polymorpha amine oxidase; LSAO, lentil seedling amine oxidase; PKAO, pig kidney amine oxidase; PSAO, pea seedling amine oxidase; TPQ, 6-hydroxydopa(2,4,5-trihydroxyphenethylamine) quinone (TOPA); TPQ aq ,Cu II -aminoquinol; TPQ sq ,Cu I –semiaminoquinolamine radical; XRD, X-ray diffraction. FEBS Journal 274 (2007) 2585–2595 ª 2007 The Authors Journal compilation ª 2007 FEBS 2585 E red þ O 2 þ H 2 O ! E ox þ NH þ 4 þ H 2 O 2 AOs are homodimers; each subunit (molecular mass @70–90 kDa) contains an active site composed of a tightly bound Cu 2+ and a quinone of 2,4,5-tri- hydroxyphenylalanine (TPQ or TOPA) [2]. Six AOs [3–8] (including a lysyl oxidase, from Pichia pastoris) have been crystallized previously, and characterized by single-crystal X-ray diffraction (XRD). The well- defined active site within these enzymes presents the following peculiar structural and functional features (Table 1): (a) TPQ is derived from the copper-cata- lyzed oxidation of a post-translationally modified tyrosine residue in the consensus sequence Asn-Tyr- Asp ⁄ Glu of the polypeptide chain [9]; (b) the copper ion is coordinated with the imidazole groups of three conserved histidine residues and with two water mole- cules (equatorial We and axial Wa) ) TPQ is close but not bound to the Cu 2+ , and appears to have high rotational mobility; (c) after the amine nucleophilic attack, the proton abstraction requires the presence of a base, which has been identified in a conserved aspar- tate residue; (d) a tyrosine residue seems to play an important role in the active site as a result of its hydrogen bond to O-4 of TPQ. Moreover, several amino acid residues have been shown to be critical in the proper positioning of TPQ during catalysis [10]. One amino acid implicated in the catalytic mechanism of some Cu⁄ TPQ AOs is a lysine residue (see below for references), although its func- tion is somewhat elusive. For example, in the recom- binant AO from Arthrobacter globiformis, during TPQ formation from the oxidation of an intrinsic tyrosine in the amino acid sequence due to a post-translational event, the copper ion catalyzes the insertion of an oxygen atom into the tyrosine ring to generate dihydroxyphenylalanine, which, upon oxidation and through the formation of the Cu I ⁄ semiquinone radical intermediate, gives rise to dihydroxyphenylalanine quinone. This observed semiquinone radical has been postulated to be covalently linked to a lysyl e-amino group of the protein [11], even though this hypothesis was ruled out by the same authors in a later paper [12]. Again in A. globiformis AO, two lysine residues, Lys184 and Lys354, situated close to the entrance of a suitable channel through which substrates and prod- ucts can access and exit the TPQ active site, have been found to be essential for the catalytic activity of the holoenzyme [12], although they do not seem to be involved in the formation of TPQ in the apoenzyme [5,12]. A nucleophilic residue has been shown with cer- tainty to be involved in the inhibition mechanism of AOs during the oxidation of 1,4-diamino-2-butyne (DABY) [13,14], 1,5-diamino-2-pentyne [15], the aro- matic monoamine tyramine [16], and other selective AO inhibitors [17]. Although the involvement of a lysine has been postulated [14,17], compelling evidence has not been presented. Finally, an important lysine has been suggested in the crystal structure of pea AO at the active site (Lys296), forming a hydrogen bond with the phe- nolic group of TPQ [4], although Duff et al. [18] later demonstrated that the published crystal showed TPQ in a nonproductive ‘on-copper conformation’. The role of Lys296 in the ‘off-copper conformation’ is therefore still unclear. The ‘on-copper and off-cop- per conformations’ refer to the orientation of TPQ and copper, as is clearly described by Dawkes & Phillips [19]. It is well known that the noble gas xenon specifically interacts with the hydrophobic interior of proteins, and an increasing number of papers in the recent lit- erature confirm that 129 Xe NMR spectroscopy is a very good technique for the characterization of cavities and channels in biologically related compounds [20–27]. Moreover, it is generally believed that xenon atoms can induce structural changes in some of the cavities or channels that they are bound to, both in solution [28] and in the solid state [29]. Xenon has been used as a probe for dioxygen-binding cavities in copper AOs by recording XRD data under pressure of xenon gas [7,18], and in a recent paper [30] we demon- strated that, under 10 atm of xenon gas, an AO from lentil seedlings can generate the free radical intermedi- ate of TPQ (TPQ sq ) in the absence of substrates, a process that probably involves a lysine residue at the active site. In this article, we investigate the binding of xenon to highly purified AOs from various sources, and our results strongly support the hypothesis that a lysine residue is implicated in the catalytic mechanism of plant enzymes. Table 1. Conserved amino acid residues in Cu ⁄ TPQ-containing AOs. Enzyme BSAO PSAO ECAO HPAO AGAO TPQ 470 387 466 405 382 Hys 519 442 524 456 431 521 444 526 458 433 683 603 689 624 592 Asp 385 300 383 319 290 Tyr 371 286 369 305 284 Lysine residue and copper–quinoproteins A. Mura et al. 2586 FEBS Journal 274 (2007) 2585–2595 ª 2007 The Authors Journal compilation ª 2007 FEBS Results 129 Xe chemical shift and spin-lattice relaxation time in AO solution In a recent paper [30], 129 Xe chemical shift and spin- lattice relaxation time studies in the presence of lentil seedling AO (LSAO) showed that the chemical shift of 129 Xe changes as a function of protein concentration (10.4 p.p.m.Æmm )1 ), and that the relaxation time (T 1 ¼ 3.2 s) is significantly reduced as compared to T 1 in the buffer ($ 500 s). These changes are commonly used as a tool to produce evidence of xenon–protein interactions [30]. In the present study, three AOs [pea seedling AO (PSAO), Euphorbia characias AO (ELAO) and pig kidney AO (PKAO)] were tested by 129 Xe NMR spectroscopy. Figure 1 shows the 129 Xe NMR spectra of the PKAO and ELAO AOs compared with the 129 Xe NMR spectra of LSAO and xenon dissolved in buffer solution. The presence of a single resonance in the protein solution indicates that xenon undergoes fast exchange in all available environ- ments. Under 10 atm of xenon gas, the 129 Xe NMR signal in AO samples is shifted downfield (ELAO 3.96 p.p.m. per 0.35 mm, corresponding to 11.3 p.p.m.Æmm )1 , and PKAO 1.4 p.p.m. per 0.15 mm, cor- responding to 9.4 p.p.m.Æmm )1 ) as compared to the resonance of the same amount of xenon in the buffer, which is used as a reference and set to 0 p.p.m. More- over, the T 1 value of all native enzymes is found to be much smaller than the T 1 value of xenon in the buffer (ELAO T 1 ¼ 4.3 ± 0.5 s; PKAO T 1 ¼ 5.5 ± 0.8 s; buffer T 1 ¼$500 s). These features, which were also observed in LSAO and other protein solutions [30], are due to the fast exchange of xenon between both speci- fic and nonspecific sites of the proteins and the buffer, and they also confirm that there is an interaction between the dissolved xenon and the interior of the protein. However, such 129 Xe NMR experiments can- not provide a more detailed characterization of the interaction between xenon and the protein, and the actual location of a possible involved cavity or cavities remains unknown and would require further studies; this, however, is beyond the purpose of this work. Owing to the high enzyme concentrations (0.25– 0.35 mm) and the low ionic strength (1 mm) of the buffer used in the experiments in the presence of xenon, we were unable to obtain significant results with bovine serum AO (BSAO), on account of its tendency to form an irreversible inactive precipitate under such experimental conditions. Xenon-induced spectroscopic features in plant enzymes Owing to the presence of the TPQ cofactor, the oxidized form of AOs has a distinctive pink color and absorbs in the visible region: BSAO shows an electronic absorption band at 476 nm (e 476 ¼ 3800 m )1 Æcm )1 ) [31], PKAO at 490 nm (e 490 ¼ 4000 m )1 Æcm )1 ) [32], PSAO and LSAO at 498 nm (e 498 ¼ 4100 m )1 Æcm )1 ) [33,34], and ELAO at 490 nm (e 490 ¼ 6000 m )1 Æcm )1 ) [35]. Addition of a substrate to a solution containing AOs in the absence of air caused the visible absorption band to disappear immediately, indicating the rapid forma- tion of a reduced TPQ intermediate, the TPQ aq , which can behave differently in plant AOs and mammalian AOs. Hence, a successive different behavior occurs. In plant AOs, TPQ aq equilibrates rapidly with the TPQ sq species by transferring one electron to copper, which is in turn reduced from the cupric to the cuprous state, and the solution immediately turns yellow as a result of the formation of new absorption bands centered at 464, 434 and 360 nm [36] (Fig. 2). In PKAO, the transfor- mation of TPQ aq to TPQ sq was observed only in the presence of CN – [37] (Fig. 2). On the other hand, BSAO, an enzyme which is not formed in the radical species during the normal catalytic cycle [38], stayed in the reduced aminoquinol form. As previously reported [30], when a solution con- taining LSAO (10 lm) was equilibrated with 10 atm of xenon gas without a substrate, after a marked lag per- iod ($ 6 h), bleaching of the 498 nm band started with Fig. 1. 129 Xe NMR spectra of AOs. 129 Xe (10 atm) spectra in a solu- tion (sodium phosphate buffer 1 m M, pH 7.0, 20% D 2 O) containing 0.35 m M ELAO, 0.28 mM LSAO and 0.15 mM PKAO. Shifts refer to the 129 Xe chemical shift in buffer. The 129 Xe NMR spectrum of PSAO, not shown, is very similar to the LSAO spectrum. A. Mura et al. Lysine residue and copper–quinoproteins FEBS Journal 274 (2007) 2585–2595 ª 2007 The Authors Journal compilation ª 2007 FEBS 2587 contemporaneous formation of TPQ sq spectral fea- tures. Similar behavior was observed with AOs from pea seedlings and E. characias latex (Fig. 3). This spe- cies reached its maximum concentration after 48 h. After readmission of oxygen, the absorption spectrum of oxidized TPQ was recovered, and approximately 1 mol of ammonia and 1 mol of hydrogen peroxide per mole of the ELAO (or PSAO) active site were detected at the end of the experiment. The results obtained with mammalian proteins were different. For PKAO, where the semiquinolamine rad- ical appears in the presence of the substrate and CN – [37], bleaching of the 490 nm band started with a marked time lag ($ 6 h) after addition of 10 atm of xenon gas (Fig. 3). It is interesting to note that the radical species formed neither in the presence nor in the absence of CN – . As observed in plant enzymes, the absorption spectrum of oxidized TPQ was recovered after readmission of oxygen, and 1 mol of ammonia and 1 mol of hydrogen peroxide per mole of active site were detected. In BSAO, no changes in the spectral features were observed under 10 atm of xenon gas, indicating that the TPQ cofactor remained in its oxidized form. Characteristics of xenon-treated AOs After exhaustive dialysis, the xenon-treated LSAO was allowed to react with a substrate under anaer- obic conditions, and behavior similar to that of the native enzyme was observed. Nevertheless, the cata- lytic activity of xenon-treated LSAO towards putres- cine was shown to be about 40% of that of the native LSAO (Table 2), whereas the k c for benzylam- ine did not change (Table 2). Also, whereas the K m values for oxygen and benzylamine were similar with native and xenon-treated LSAO, the K m for the amine putrescine was considerably higher (Table 2). The k c ⁄ K m ratio, a more useful measure of substrate specificity, was shown to be dramatically reduced, and a comparison with those obtained for other AOs is shown in Table 2. Very similarly to LSAO, loss in activity was also seen in PSAO and in ELAO. The catalytic activity of xenon-treated PKAO towards cadaverine and benzylamine was shown to be about 20% of that of the native enzyme (Table 2). Xenon-treated BSAO, which retains its oxidized form, showed the same activity as the cor- responding native enzyme (Table 2). 600500400300 0.3 0.2 0.1 0 Wavelength (nm) Absorbance Absorbance A 600500400300 0.15 0.1 0.05 0 Wavelen g th (nm) B Fig. 3. Absorption spectra changes of ELAO and PKAO native enzyme under 10 atm of xenon gas. Conditions: (A) ELAO, 11 l M, and (B) PKAO, 19 lM,in1mM sodium phosphate buffer (pH 7.0). The spectra of the reduced forms (–––) were recorded after 48 h. 600500400300 0.2 0.15 0.1 0.05 0 Wavelength (nm) AbsorbanceAbsorbance A 600500400300 0.2 0.1 0 Wavelen g th (nm) B Fig. 2. Absorption spectra of AOs. (A) Native LSAO, 16 lM,in 1m M sodium phosphate buffer (pH 7.0), under anaerobic condi- tions before (- - -) and after (–––) addition of 10 m M putrescine. (B) PKAO, 19 l M,in1mM sodium phosphate buffer (pH 7.0), before (- - -) and after (–––) addition of 10 m M cadaverine in anaerobic con- ditions and in the presence of 100 l M CN – . Lysine residue and copper–quinoproteins A. Mura et al. 2588 FEBS Journal 274 (2007) 2585–2595 ª 2007 The Authors Journal compilation ª 2007 FEBS Oxidative deamination of a lysine residue The oxidative deamination of a lysine residue was monitored through the formation of a-aminoadipic-d- semialdehyde-derivatized fluoresceinamine by HPLC (see Experimental procedures) [39]. As reported previ- ously [30], with xenon-treated native LSAO, 1 mol of allysine residue per mole of monomeric enzyme was detected. Identical results were obtained for PSAO, ELAO, and PKAO, whereas with BSAO, where no reduction occurred, no allysine residue was detected. Reaction with the mechanism-based inhibitor The experimental findings clearly show that plant and mammalian AOs under 10 atm of xenon are reduced without the presence of an amine substrate. In the presence of xenon, plant enzymes form yellow TPQ sq , whereas in pig enzyme the bleached species TPQ aq is observed. A lysine residue at the active site may be implicated in this mechanism. An important method in studying the structure–function of an enzyme is to find specific inhibitors and follow their effects. Our interest in the present study is in the mechanism-based inhib- itor DABY, for the following two reasons: (a) the inhibitor has been found to be a suicide substrate for plant copper AO (Cu-AO) from pea seedlings [14] and grass pea [15], and for mammalian AOs from pig kid- ney [40] and from beef serum [17]; and (b) it has been postulated that the irreversible inhibition of all enzymes involves an intermediate aminoallenic com- pound that forms covalently bound pyrrole in the reac- tion with a nucleophile at the active site. The exact mechanism of inhibition was elusive, and it was only in grass pea AO that the involved nucleo- phile was identified as Glu113, a residue corresponding to a Lys113 in PSAO [14]. DABY was also shown to be a mechanism-based inactivator for native LSAO and ELAO, with a k inh of 0.1 min )1 and a half-max- imal inactivation of 4 · 10 )5 m for ELAO (Fig. 4), and a k inh of 5 min )1 and a half-maximal inactivation of 4 · 10 )4 m for LSAO. Moreover, all the xenon-trea- ted AOs were inactivated by the reaction with DABY, clearly indicating that the lysine residue involved in the reduction of TPQ under xenon pressure is not the nu- cleophilic residue involved in the DABY inhibition mechanism; that is, the reactive turnover product of DABY binds an amino acid residue without interfering with the TPQ function. Discussion In the past decade, several interesting reports have been published on the catalytic mechanism of AOs, and a significant number of essential amino acid resi- dues have been identified by site-specific mutagenesis. In this article, we show that a lysine is an important residue and that it plays a key role in modulating the activity of plant AOs, as in the mammalian AO from pig kidney, and we tentatively assign this role to a lysine at the active site. Table 2. Kinetic parameters of Cu ⁄ TPQ-containing AOs. Enzyme k c s )1 K m (mM) k c ⁄ K m LSAO 155 a 0.24 646 1 b 0.45 2.2 Xenon-treated 62 a 1.4 44.2 LSAO 0.9 b 0.46 1.95 ELAO 38 b 0.2 190 0.18 b 0.4 0.45 Xenon-treated 13.3 a 1.9 7 ELAO 0.17 b 0.4 0.43 PSAO 140 a 0.2 700 0.5 b 0.45 1.1 Xenon-treated 53.2 a 1.5 35.5 PSAO 0.5 b 0.45 1.1 BSAO c 1 d 0.1 10 0.35 b 2.2 0.16 PKAO 4.5 e 0.1 45 0.23 b 0.12 1.91 Xenon-treated 0.9 e 0.23 3.9 PKAO 0.046 b 0.25 0.18 a Using putrescine as substrate. b Using benzylamine as substrate. c In BSAO, there are no differences in the kinetic parameters before and after xenon treatment (not shown). d Using spermine as substrate. e Using cadaverine as substrate. SDs are not reported. 3020100 100 10 Time (min) Residual activity (%) 0.090.060.030 40 20 0 1/[DABY] (µ M -1 ) k/1 p pa Fig. 4. Inactivation of ELAO by DABY. The enzyme (6 nM) was pre- incubated with the indicated concentrations of DABY at 25 °Cin 1m M sodium phosphate buffer (pH 7.0). The concentrations of DABY were: d,10l M; s,20lM; .,30lM; w,40lM. Inset. Dou- ble reciprocal plot of apparent first-order rate constants of inactiva- tion (k app ) vs. DABY concentrations. A. Mura et al. Lysine residue and copper–quinoproteins FEBS Journal 274 (2007) 2585–2595 ª 2007 The Authors Journal compilation ª 2007 FEBS 2589 All plant AOs used in the present study contain 38 lysines in each subunit (Protein Data Bank accession numbers: ELAO AF171698; PSAO L39931; LSAO X64201). Because, as reported previously [30], the elu- tion profiles resulting from HPLC analysis of the AO proteolytic digestion with trypsin and lysyl endopepti- dase are very complicated, it is extremely difficult to determine with certainty which lysine residue is conver- ted into allysine. As the identity in amino acid sequences of ELAO, LSAO and PSAO is about 92%, it would be safe to accept that both enzymes have an almost identical structure; that is, the three enzymes could have two identical subunits, each containing three structural domains (D2, D3, and D4). As observed in the crystal structure of PSAO, copper ion and TPQ are in close proximity (shorter distance $ 6A ˚ ) [4], but they are not coordinated. Moreover, a slight displacement of TPQ would be required to facili- tate the extremely fast electron transfer between TPQ aq and TPQ sq , and the TPQ side chain appears suffi- ciently flexible to accommodate such a change. Although TPQ has been found to be characterized by considerable conformational flexibility, it has also been pointed out that when an amine substrate attacks the TPQ at C-5, H + abstraction of the active site base Asp300 would require it to rotate by 180° [4]. This sig- nificant displacement would contrast with the previous observation that the TPQ cofactor could remain fixed during the catalytic cycle [41–43]. Currently, new forms of PSAO native protein crystal are available [18] in the so-called ‘off-copper conformation’. In this structure, the O-4 of TPQ is hydrogen bonded to the hydroxyl group of conserved tyrosinyl residue Tyr286, and the TPQ orientation is in the active form, with the aspartic active site base residue (Asp300) in an excellent posi- tion for abstraction of the Ca proton from the sub- strate, so that TPQ does not rotate during the catalytic mechanism. Duff et al. have recently reported a new crystal form of the P. pastoris lysyl oxidase that has a covalent crosslink between two lysine residues, Lys778 and Lys66 [44]. Whereas Lys778 can readily reach the TPQ cofactor in the active site of the enzyme without any other conformational changes, Lys66 is in a well- ordered region and cannot do so. The authors pro- posed that the lysyl oxidase oxidized Lys778 to the corresponding aldehyde allysine, which can react spon- taneously with Lys66, which is is nearby and appropri- ately oriented. X-ray crystallography of PSAO has also demonstra- ted that a lysine residue, Lys296, is located in domain D4, between b-sheet C-5 and a helix H-8, close to the entrance to a channel found to be suitable for moving substrate and products to and from the copper ⁄ TPQ active site buried in the protein interior. This residue forms a hydrogen bond with the phenolic group of TPQ when in a nonproductive ‘on-copper conforma- tion’ [4], but its role in the ‘off-copper conformation’ is still unknown. This amino acid is conserved in LSAO (Lys296) and ELAO (Lys302) (Fig. 5). In BSAO (Protein Data Bank accession number S69583), the residue corresponding to Lys296 in LSAO is Thr381 (Fig. 5), but an arginine is present at position 382. Although the amino acid sequence of PKAO is unknown, there may be a threonine residue, as in human kidney AO (Thr369), considering its great homology with known reported sequences [45] (Fig. 5). In this case, a lysine (Lys370) flanks the threonine resi- due that could react with TPQ. This is evidence for the importance of a lysine residue in the active site for the formation of the radical species in plant enzymes and the aminoquinol in PKAO under xenon pressure. As the arginine residue in BSAO possesses a highly basic guanidine group, it could be unreactive with TPQ under xenon pressure. Fig. 5. Partial amino acid sequence alignment of some AOs. The active site base aspartate residue is in yellow; the lysine residue at the active site, probably involved in the formation of the radical in plant AOs, is in green; the nucleophilic residue probably involved in the mechanism- based inhibition by DABY is in red. The Gene Bank accession numbers of each sequence are: PSAO, AB026253; LSAO, X64201; ELAO, AF171698; and BSAO, S69583. HKAO (human kidney AO) is from Novotny et al. [45]. Lysine residue and copper–quinoproteins A. Mura et al. 2590 FEBS Journal 274 (2007) 2585–2595 ª 2007 The Authors Journal compilation ª 2007 FEBS Another interesting result is that xenon-treated LSAO shows lower activity and a higher K m value for diamine putrescine as substrate, but not for aromatic monoamine benzylamine. However, xenon treatment of PKAO was accompanied by loss of activity for both cadaverine and benzylamine. These results are most compatible with two different mechanisms being involved in the interaction between enzyme and sub- strate. It is possible, only in the plant enzyme, that the e-amino group of Lys296 may interact with the posit- ive charge of the amino group of putrescine, as shown in Fig. 6. This residue could have an important role in conferring substrate specificity, with consequences for catalytic efficiency when lysine is transformed into allysine. Thanks to DABY, a mechanism-based inhibitor, we can confirm that an amino acid residue is impli- cated in the mechanism-based inhibition that is dif- ferent from the residue implicated in TPQ reduction under xenon pressure. A nucleophile residue is implicated in the DABY inhibition mechanism, and as reported by Fre ´ bort et al. [14], the Lys113 in PSAO could be implicated in the formation of pyrrole. This residue could correspond to Asp113 in LSAO, Asp117 in ELAO, and Asp179 in BSAO (Fig. 5). In a recent paper, three Cu-AOs [Arthrobacter globiformis amine oxidase (AGAO), PSAO and P. pastoris lysyl oxidase] were investigated by Duff et al. [18] by XRD under high xenon pressure, with the aim of finding a potential dioxygen-binding cavity close to the active site of Cu-AO that is related to enzyme function. In all three xenon derivatives, the xenon proved to be bound at a variety of cavities and with a range of occupancies. The xenon sites closest to the Cu ⁄ TPQ center in each structure are: Xe–Cu % 7.5 A ˚ and Xe–TPQ % 9.5 A ˚ . From this study, the authors concluded that the results do not give enough evidence of a xenon-binding site in a region of the molecule close to the active site to justify the suggestion of a potential transient dioxygen-binding site. In addressing the usefulness of 129 Xe NMR spectros- copy in the characterization of biological compounds in solution, it must be pointed out that these systems are generally characterized by complex structures and often by the presence of more than one specific site for ligands and ⁄ or substrates. The nearest neighbor resi- dues of the bound xenon atoms in the cavities are pre- dominantly nonpolar side chains, but they include polar side chains and backbone peptide groups. This, together with the fact that the observed 129 Xe chemical shift is dynamically averaged among different binding sites and at the same time interacts with the protein surface, makes it difficult to separate the individual contributions so as to show whether a particular xenon-binding site is responsible for the different com- ponents observed in the studied AOs in solution. Hyperfine interactions with unpaired electrons in radical species and ⁄ or paramagnetic metal ions could be a further source of information, as long as they can be distinguished from other structural or dynamic fac- tors affecting NMR parameters. These 129 Xe NMR outputs cannot provide local information on the host–guest interaction involved. Experimental evidence of the fast diffusion of xenon within AOs clearly opposes the static and average pic- tures given by single-crystal XRD structures, which seem to show that xenon atoms are localized at specific sites. Moreover, it is worth noting that, as the single- crystal XRD results utterly ignore the fundamental dynamic features involved in the functionality of the biomolecules in solution, hypotheses on biological activities based on crystal structures should be consi- dered critically. Fig. 6. Active site of plant AOs. The model of the active site shows the possible interaction with two substrates: benzylamine, which represents a substrate with an apolar chain, and putrescine, with a positively charged amino group. The positively charged e-amino group of lysine exerts a repulsive force towards substrates charac- terized by the presence of a positively charged amino group, such as putrescine, leading to a lower catalytic efficiency when lysine is transformed into allysine. Neither lysine nor allysine can interact with the apolar chain of benzylamine, leading to this amino acid residue being responsible for the different substrate specificities. A. Mura et al. Lysine residue and copper–quinoproteins FEBS Journal 274 (2007) 2585–2595 ª 2007 The Authors Journal compilation ª 2007 FEBS 2591 Concluding remarks The TPQ sq radical represents the highly reactive spe- cies with the oxygen molecule in the catalytic cycle of plant AOs. Thus, the radical species observed under 10 atm of xenon without a substrate in plant AOs only, and the fact that lysine was identified at the act- ive site, could reveal key aspects of the structure–func- tion relationship among various AOs. Moreover, plant enzymes show a high affinity for putrescine and a lower activity for benzylamine. In contrast, xenon-trea- ted plant AOs show a high loss in catalytic activity towards putrescine, but not towards benzylamine. The transformation of a lysine residue, probably Lys296, into allysine, four residues from the active site base identified in a conserved aspartate residue (Asp300), could have an important role in the recognition of sub- strates with a positively charged amino group. In conclusion, although the data reported in the pre- sent article may well be valid generally, the exact loca- tion and nature of the observed interactions between xenon and the enzymes studied remain somewhat hypothetical and are not of any functional significance. Nevertheless, from our results, we conclude that xenon is capable of forcing a conformational change in AOs, such that most of them react with one of their own lysine residues. As reported for other amino acid resi- dues, changes in active site architecture and charge dis- tribution seem to be critical during catalysis in AOs. Thus, further comparative investigation of the active site in AOs from plants, mammals and bacteria is nee- ded to understand whether these enzymes, which differ in structure and action mechanism, follow a similar metabolic pathway. Experimental procedures Materials All reagents were of the highest purity degree available. 1,4-Diaminobutane dihydrochloride (putrescine), 1,5-dia- minopentane dihydrochloride (cadaverine), benzylamine hydrochloride and N,N¢-bis(3-aminopropyl)-1,4-butane diamine tetrahydrochloride (spermine) were purchased from Sigma Aldrich (St Louis, MO). Xenon chemical shift meas- urements were made using 92% enriched 129 Xe (Chemical Research 2000; Rome, Italy). DABY was synthesized as previously reported [13]. Enzymes AOs from bovine plasma (BSAO; k c ¼ 0.35 s )1 using ben- zylamine as substrate) [31], pig kidney (PKAO; k c ¼ 4.5 s )1 using cadaverine as substrate) [32], pea seedlings (PSAO; k c ¼ 140 s )1 using putrescine as substrate) [33], lentil seed- lings (LSAO; k c ¼ 155 s )1 using putrescine as substrate) [34] and E. characias latex (ELAO; k c ¼ 23 s )1 using putrescine as substrate) [35] were prepared according to the described procedures. The activities of the tested enzymes were measured according to the procedures reported in the related refer- ences. Oxygen uptake was determined with a Clark-type electrode coupled to an OXYG1 Hansatech oxygraph (Hansatech Instruments Ltd, King’s Lynn, UK). The tem- perature of the reaction chamber was kept at 37 °Cby using a circulating water bath. The solution (1 mL) con- taining the enzyme in a 1 mm sodium phosphate buffer (pH 7.0) was maintained for 20 min at a constant level of oxygen, as previously reported [46,47], and the reaction was started by addition of the related substrate. The K m values for AOs using different substrate concentrations at a satur- ating concentration of oxygen (219 lm), or varying concen- trations of oxygen at a saturating concentration of substrate, were calculated from initial velocity data fitted to the Michaelis–Menten equation by nonlinear regression and by double reciprocal plots by Michaelis–Menten analysis in a1mm sodium phosphate buffer (pH 7.0). Benzylamine oxidase activity was measured in a 1 mm sodium phosphate buffer (pH 7.0), by monitoring the increase in absorbance of UV light at 250 nm using an e 250 ¼ 12.8 mm )1 Æcm )1 for benzaldehyde [36]. Catalytic center activity (k c ) is defined as mole of substrate consumed per mole of active sites · s )1 . Spectroscopic methods UV ⁄ visible experiments Absorption spectra of AOs in a 1 m m sodium phosphate buffer (pH 7.0) were recorded at 25 °C with an Ultrospec 2100 spectrophotometer (Biochrom Ltd, Cambridge, UK). Anaerobic experiments were performed with a Thunberg- type spectrophotometer cuvette (Soffieria Vetro, Sassari, Italy). Solutions were subjected to several cycles of evacu- ation followed by flushing with argon. 129 Xe NMR experiments Experiments were carried out as previously reported [20]. Briefly, samples of native AOs in a 1 mm sodium phos- phate buffer (pH 7.0), with 20% D 2 O, were degassed using three freeze–pump–thaw cycles, pressurized with 10 atm of xenon gas into Wilmad high-pressure NMR tubes (outside diameter 5 mm and internal diameter 7.1 mm; outside diameter 5 mm and internal diameter 2.2 mm; Buena, NJ) and allowed to equilibrate for 48 h. 129 Xe NMR spectra were recorded on a Varian VXR-300 spectrometer (Varian, Palo Alto, CA), and 129 Xe NMR spin lattice relaxation times (T 1 ) of native AOs were measured using the inversion Lysine residue and copper–quinoproteins A. 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An important lysine residue in copper ⁄ quinone-containing amine oxidases Anna Mura 1 , Roberto Anedda 2 , Francesca Pintus 1 , Mariano Casu 2 , Alessandra Padiglia 1 , Giovanni Floris 1 and. leading to a lower catalytic efficiency when lysine is transformed into allysine. Neither lysine nor allysine can interact with the apolar chain of benzylamine, leading to this amino acid residue. monoamine tyramine [16], and other selective AO inhibitors [17]. Although the involvement of a lysine has been postulated [14,17], compelling evidence has not been presented. Finally, an important