Two L-amino acid oxidase isoenzymes from Russell’s viper (Daboia russelli russelli) venom with different mechanisms of inhibition by substrate analogs Somnath Mandal and Debasish Bhattacharyya Division of Structural Biology and Bioinformatics, Indian Institute of Chemical Biology, Kolkata, India Keywords enzyme kinetics; inhibitor cross-competition; flavoenzyme; L-amino acid oxidase; mixed inhibition Correspondence D Bhattacharyya, Division of Structural Biology and Bioinformatics, Indian Institute of Chemical Biology, Kolkata 700032, India Fax: 91 33 2473 5197 ⁄ 0284 Tel: 91 33 2473 3491 x 164 E-mail: debasish@iicb.res.in (Received January 2008, revised 25 February 2008, accepted 27 February 2008) doi:10.1111/j.1742-4658.2008.06362.x Two isoforms, L1 and L2, of l-amino acid oxidase have been isolated from Russell’s viper venom by Sephadex G-100 gel filtration followed by CMSephadex C-50 ion exchange chromatography The enzymes, with different isoelectric points, are monomers of 60–63 kDa as observed from size exclusion HPLC and SDS ⁄ PAGE Partial N-terminal amino acid sequencing of L1 and L2 showed significant homology with other snake venom l-amino acid oxidases Both the enzymes exhibit marked substrate preference for hydrophobic amino acids, maximum catalytic efficiency being observed with l-Phe Inhibition of L1 and L2 by the substrate analogs N-acetyltryptophan and N-acetyl-l-tryptophan amide has been followed The initial uncompetitive inhibition of L1 followed by mixed inhibition at higher concentrations suggested the existence of two different inhibitor-binding sites distinct from the substrate-binding site In the case of L2, initial linear competitive inhibition followed by mixed inhibition suggested the existence of two nonoverlapping inhibitor-binding sites, one of which is the substratebinding site An inhibition kinetic study with O-aminobenzoic acid, a mimicking substrate with amino, carboxylate and hydrophobic parts, indicated the presence of three and two binding sites in L1 and L2, respectively, including one at the substrate-binding site An inhibitor cross-competition kinetic study indicated mutually excluding binding between N-acetyltryptophan, N-acetyl-l-tryptophan amide and O-aminobenzoic acid in both the isoforms, except at the substrate-binding site of L1 Binding of substrate analogs with different electrostatic and hydrophobic properties provides useful insights into the environment of the catalytic sites Furthermore, it predicts the minimum structural requirement for a ligand to enter and anchor at the respective functional sites of LAAO that may facilitate the design of suicidal inhibitors The flavoenzyme l-amino acid oxidase (LAAO; EC 1.4.3.2) is a major constituent of many snake venoms This enzyme catalyzes oxidative deamination of l-amino acid substrates to a-keto acids in a stereospecific mode The catalytic cycle begins with the reductive half-reaction involving the conversion of FAD (flavin cofactor) to FADH2 and concomitant oxidation of the amino acid to an imino acid The imino acid intermediate of the oxidation pathway undergoes nonenzymatic hydrolysis to yield the respective a-keto acid and ammonia An oxidative half-reaction completes the catalytic cycle by reoxidizing FADH2 Abbreviations LAAO, L-amino acid oxidase; NAT, N-acetyltryptophan; NATA, N-acetyl-L-tryptophan amide; OAB, O-aminobenzoic acid; PBE, polybuffer exchanger; PLA2, phospholipase A2; RVV, Russell’s viper venom 2078 FEBS Journal 275 (2008) 2078–2095 ª 2008 The Authors Journal compilation ª 2008 FEBS Inhibitor-binding sites of L-amino acid oxidase S Mandal and D Bhattacharyya NH+ NH3 R C H Scheme Reaction mechanism of LAAO COO– R COO– R C COO– NH3 E-FAD E-FADH2 O2 H2O2 with molecular oxygen, producing hydrogen peroxide (Scheme 1) The redox catalytic cycle of the enzyme has been well documented in the literature [1–3] The preferred substrates of these enzymes are aromatic or, more generally, hydrophobic amino acids Deamination of polar and basic amino acids proceeds at a much lower rate [3,4] So far, LAAOs from bacterial, fungal and plant sources have been proposed to be involved in the utilization of nitrogen sources [5,6] The role of this enzyme in snake venom is not fully understood Venom LAAOs act as potential toxins, as they cause impairment of platelet aggregation together with induction of necrotic and apoptotic cell death [4] The cytotoxicity of LAAO is primarily attributed to hydrogen peroxide produced during the substrate turnover [7,8] Recently, LAAO from the Malayan pit viper has been shown to induce both necrosis and apoptosis in Jurkat cells, where the role of hydrogen peroxide was well established by scavenging it with catalase Docking of LAAO on the cell surface and subsequent internalization has been proposed to be the inherent mechanism for induction of apoptosis [9] The evidence to date implicates the glycan moiety of LAAO for the docking onto the host cell that enhances localization of high concentrations of hydrogen peroxide The mode of hydrogen peroxide delivery has been suggested to be an important factor for efficient and tissue-specific induction of apoptosis [7,10] Apart from mechanistic studies, the crystal structure of LAAO from Calloselasma rhodostoma complexed ˚ with l-Phe has been recently solved at 1.8 A resolution [11] It predicted deprotonation of the a-NH3+ group of the substrate by His223 of the enzyme and subsequent movement of the lone pair of electrons from NH2 to the a-C atom, which activates the substrate to transfer the hydride from a-C to N-5 of the flavin moiety to yield the imino acid and FADH2 [11] The structure of the catalytic site of LAAO in complex with two different inhibitors indicates that the site is buried deep inside the molecule [12] Each subunit of the dimeric enzyme is composed of three parts: an FAD- C O H2O binding domain, a substrate-binding domain, and a helical domain The interface between the substrate˚ binding and helical domains forms a 25 A long funnel providing access to the active site Binding sites and orientations of O-aminobenzoic acid (OAB) (a substrate analog without the a-CH that is necessary for hydride transfer) within the catalytic funnel suggest the probable role of electrostatic and hydrophobic parts in the trajectory of the substrate to the active site However, the importance of complementary electrostatic and hydrophobic surfaces in the inhibitor molecule for successful inhibition of enzymatic activity remains to be evaluated Envenomation by Russell’s viper followed by death is a WHO-identified occupational hazard for paddy growers of Southeast Asian countries [13] Currently, application of antivenom is the only mode of treatment, and the success rate varies to a large extent For better snakebite management, it is our long-term aim to isolate and characterize different toxins These include hemorrhagins such as VRR-22 [14], VRR-12 [15], VRR-76 [16] and Russell’s viper venom (RVV)-7, a cytotoxin with phospholipase A2 (PLA2) activity [17] that also acts as a renal tubular necrosis factor [18] Empirical assay protocols for hemorrhage and PLA2 activity that are suitable for snake venoms have also been developed [19,20] Here we report the purification and preliminary characterization of two LAAO isoforms from RVV and demonstrate for the first time that they contain two or three inhibitor-binding sites in solution Derived N-terminal sequences of the two isoforms have shown a good degree of homology to LAAO from other snake venoms A recent molecular and comparative structural analysis of Bothrops jararacussu and Bothrops moojeni having 83–87% sequence identities with other snake venom LAAOs indicated a high degree of structural similarity in the main regions, such as the FAD-binding, substrate-binding and helical domains, with those of Ca rhodostoma LAAO [21] Our observations regarding the inhibition kinetics of LAAO from RVV were compared with the crystal structure of Ca rhodostoma LAAO, based on similar FEBS Journal 275 (2008) 2078–2095 ª 2008 The Authors Journal compilation ª 2008 FEBS 2079 Inhibitor-binding sites of L-amino acid oxidase S Mandal and D Bhattacharyya using an NaCl gradient of 0–0.1 m at pH 7.2 (Fig 1B) Bound LAAO activity was released near the end of the gradient in two partially overlapping fractions They were collected (the middle fractions were discarded), and termed L1 and L2 Homogeneity of the fractions was demonstrated by SDS ⁄ PAGE followed by silver staining, where they appeared as single bands of equal migration (Fig 1B, inset) Development of the gel with glycoprotein staining solution appeared to be positive (result not shown) Final recoveries of LAAO activity in L1 and L2 were 22% and 18%, respectively Separation of the two isoforms into two peaks by CM-Sephadex C-50 cation exchange chromatography indicated substrate specificity and number of inhibitor-binding sites The insights gained are likely to be useful in designing suicidal inhibitors in future [22] Results Purification and characterization of LAAO Crude RVV was resolved into four major peaks on a Sephadex G-100 gel filtration column, LAAO activity being eluted in the first peak (Fig 1A) Recovery of activity was 80% The pooled portion was further fractionated by CM-Sephadex C-50 chromatography, A C B D Fig Purification and characterization of L1 and L2 from RVV (A) Chromatogram showing elution of LAAO from Sephadex G-100 (117 · 1.2 cm) column Twenty-one milligrams of RVV, equivalent to 15 mg of protein was applied Elution of protein was followed at 280 nm and LAAO by a coupled assay at 436 nm using L-Phe as substrate Fractions with LAAO activity were pooled and applied to a CMSephadex C-50 column (2 · 10 cm) pre-equilibrated with 20 mM potassium phosphate buffer (pH 7.4) (B) Bound fractions were eluted after application of 0–0.1 M NaCl (dotted line) Elution of proteins and of LAAO were followed as stated earlier Indicated areas of L1 and L2 peaks were pooled Inset: 10% SDS ⁄ PAGE profiles of the pooled L1 and L2 fractions, showing a single band corresponding to 60 kDa with respect to standard molecular mass markers, the positions of which have been indicated on the left (C) Chromatofocusing of the LAAO activity eluted from a Sephadex G-100 column The sample was loaded on a PBE 96 column pre-equilibrated with 25 mM Tris ⁄ acetate (pH 8.3) The sample was eluted with 0.0072 mmolỈpH unit)1ỈmL)1 PBE 96 (pH 6) at a flow rate of 12 mLỈh)1 LAAO activity and the pH of each 1.5 mL fraction were measured separately (D) Size exclusion HPLC of L1 and L2 using a Waters Protein Pak 300 column (fractionation range 10–400 kDa) The flow rate was 0.8 mLỈmin)1 Elution of L1 and L2 at 9.01 ± 0.05 and 9.23 ± 0.06 are marked Inset: calibration curve of the column, using standard molecular mass markers as described in the text Upward and downward arrows indicate the positions of L1 and L2, respectively 2080 FEBS Journal 275 (2008) 2078–2095 ª 2008 The Authors Journal compilation ª 2008 FEBS Inhibitor-binding sites of L-amino acid oxidase S Mandal and D Bhattacharyya different pI values The difference was further analyzed by chromatofocusing of the LAAO from Sephadex G100 chromatography This resulted in the separation of LAAO activity into two peaks corresponding to pH 7.49 ± 0.06 and pH 7.26 ± 0.04 (Fig 1C) As compared to nonglycosylated proteins, the peaks are broader but not diffused to a large extent A low level of glycosylation and homogeneity of the glycosydic part, as has been reported in the case of Ca rhodostoma LAAO [23], might be the cause of the chromatofocusing features for both the isoforms Molecular mass Purified L1 and L2 appeared in SDS ⁄ PAGE with a molecular mass corresponding to 60 kDa by reference to standard markers In size exclusion HPLC, L1 and ˚ L2 were eluted from a Protein Pak 300 A column as single symmetrical peaks at 9.01 ± 0.05 and 9.23 ± 0.06 min, respectively These corresponded to 63 and 60 kDa by reference to a calibration curve (Fig 1D) Thus, the enzymes appeared to exist as monomers under the conditions of storage N-terminal sequencing Derived amino acid sequences up to the 20th residue from N-termini of L1 and L2, obtained using Edman degradation, were ADDINPKEECFFEDDYYEFE and ADDKNPLEECFCEDDDYCEG, respectively These sequences are 70% homologous to each other Chromatograms of the released amino acid derivatives indicated that the analyzed samples were homogeneous and free from cross-contamination Homology analysis of these sequences using the NCBI blastp program indicated up to 93% similarity with LAAO from other snake venoms (Table 1) Both L1 and L2 have shown more than 60% homology with LAAO from Ca rhodostoma, for which an X-ray crystallographic structure is available This sequence similarity indicates probable structural and functional similarity between the enzymes Table Sequence homology of L1 and L2 (Daboia russelli russelli) with LAAOs from other snake venom sources Swiss Prot entry names of respective LAAOs are presented in parentheses Homology (%) LAAO source organism L1 L2 Vipera berus berus (OXLA_VIPBB) Gloydius blomhoffi j(OXLA_AGKHA) B jararcassu (OXLA_BOTJR) B moojeni (OXLA_BOTMO) Macrovipera labetina (OXLA_VIPLE) Crotalus durissus (OXLA_CRODC) Gloydius halys (OXLA_AGKHP) Crotalus atrox (OXLA_CROAT) Crotalus adamanteus (OXLA_CROAD) Ca rhodostoma (OXLA_AGKRH) 78 73 73 73 78 68 68 68 68 68 93 80 80 80 93 73 73 73 73 64 tively The spectral features of the dissociated cofactor were similar to those of FAD The shifting of absorption maxima of the enzyme-bound flavin was due to its microenvironment The RP-HPLC profiles of the cofactor dissociated by heat and of reference FAD are shown in Fig 2B This illustrates that, under the chromatographic conditions used, a single component was eluted at 10.59 ± 0.07 in either case Extension of the gradient up to 100% methanol failed to elute any additional component from the enzyme extract The HPLC fractions of reference FAD and the cofactor were collected and analyzed by ESI MS The abundance of FAD intact ions (830.32 Da) was only 20%, whereas a 413.35 Da [riboflavin, C17H20N4O6 (376.36) + K+ (39)]+1 peak appeared with 70% abundance The mass spectrum of the cofactor did not produce any peak corresponding to 830 Da, but signals of m ⁄ z 415 and 317.2 [C15H16N4O4 (316.12) + H+]+1 were present in greater abundance These peaks might have resulted from the fragmentation of intact FAD during ionization Quantification of enzyme-bound FAD was done from the absorption spectrum of the dissociated cofactor (e462 = 1.14 · 104 m)1 cm)1) The derived stoichiometry was 1.25 ± 0.03 ⁄ monomer In a control set, it was confirmed that the presence of 0.1% SDS did not affect the spectrum of FAD Identification of cofactor The absorption spectra between 300 and 600 nm of holo-LAAO from the active fractions of Sephadex G100 chromatography containing a mixture of L1 and L2, the isolated cofactor after dissociation from the enzyme and standard FAD are shown in Fig 2A In each spectrum, two peaks of comparable intensity were observed The corresponding maxima were at 390 and 475 nm, 375 and 450 nm, and 370 and 450 nm, respec- Enzymatic properties L1 and L2 have shown substrate preferences for hydrophobic, particularly aromatic, amino acids, of which l-Phe was the best Table summarizes the catalytic efficiency, i.e the ratio of turnover number and Km, of L1 and L2 for different substrates The specific activities of L1 and L2 with l-Phe were found to be 8.96 ± 1.88 and 6.94 ± 1.25 lmolỈmin)1Ỉmg)1, respectively FEBS Journal 275 (2008) 2078–2095 ª 2008 The Authors Journal compilation ª 2008 FEBS 2081 Inhibitor-binding sites of L-amino acid oxidase S Mandal and D Bhattacharyya A 90 0.006 B 10.59 ± 0.07 0.09 60 0.002 30 A450 nm Absorbance 0.004 0.05 0.03 % Methanol 0.07 0.01 0.00 350 500 400 450 Wavelength (nm) 600 0.00 5.00 10.00 Time (min) 15.00 20.00 Fig Characterization of flavin cofactor (A) UV–visible absorption spectrum between 300 and 600 nm of enzyme-bound cofactor of: (1) the sample obtained from gel filtration chromatography; (2) cofactor separated from the enzyme after heat denaturation in the presence of SDS; and (3) standard FAD (B) RP-HPLC profiles of: (1) standard FAD; and (2) cofactor extracted from the enzyme after gradual heating Fractions were eluted only in a 15–75% methanol gradient developed between and 20 A description of the experiment is provided in the text Table Substrate specificity of L1 and L2 ND, not detectable Substrate Hydrophobicitya Surface ˚ area (A2)b L-Phe +2.8 )1.3 )0.9 +1.9 +3.8 +4.5 +4.2 +1.8 210 230 255 185 170 175 155 115 L-Tyr L-Trp LMet L-Leu L-Ile L-Val L-Ala a Hydrophobicity indices [45] b L1 L2 66.5 52.0 210.9 297.3 750.8 1.44 ND ND · · · · · · 10)6 10)6 10)6 10)6 10)6 10)3 49.3 538.2 235.1 222.8 599.7 1.89 ND ND L1 · · · · · · 10)6 10)6 10)6 10)6 10)6 10)3 L2 14.06 12.62 6.41 5.68 4.32 0.88 – – 15.17 1.13 5.51 7.14 2.03 0.65 – – Accessible surface area for residues as part of a polypeptide chain [42] The catalytic efficiency of L1 on l-Tyr was close to that on l-Phe, but L2 oxidized l-Tyr with poor catalytic efficiency The hydrophobic amino acids with sur˚ face area smaller than 185 A2 acted as poor substrates, and catalytic efficiencies with amino acids of surface ˚ area £ 155 A2 were not detectable On the other hand, l-Trp, with a hydrophobicity index between those of l-Phe and l-Tyr but with a lower surface area ˚ (255 A2), had turnover efficiencies 2.2-fold and 2.8-fold less than that of l-Phe in the cases of L1 and L2, respectively Amino acids with polar or charged side chains are excluded from Table 1, as their oxidation was not detectable pH and thermal stability High-pH inactivation, as observed for LAAO from Calloselasma adamenteus [24], was assessed for L1 and 2082 Catalytic efficiency (mol)1Ỉs)1) · 104 Km (M) L2 after exposing them to pH 4.2–10 for h at 25 °C Both enzymes retained 100% activity between pH 6.5 and pH 8.8 Drastic inactivation was observed above and below this range The enzymes were exposed to different temperatures between 30 °C and 100 °C for 10 to determine their thermal stability Both were stable up to 50 °C but were sharply inactivated above 60 °C Selection of inhibitors Interaction of L1 and L2 with substrate analogs was investigated, with the expectation that the kinetic analysis would reflect the inhibitor trajectory in the functional molecule Crystallographic data indicated that the hydrophobic and electrostatic parts of the ligand might play crucial roles in the orientation and binding of the molecules in the catalytic funnel of the enzyme FEBS Journal 275 (2008) 2078–2095 ª 2008 The Authors Journal compilation ª 2008 FEBS Inhibitor-binding sites of L-amino acid oxidase S Mandal and D Bhattacharyya Table Summary of inhibition constants of L1 and L2 ND, not detected, as inhibition constants for three inhibitor-binding sites cannot be determined from kinetic data NA, not applicable L2 L1 KI (lM)a KIS (lM)b KI (lM) KIS (lM) NA NA NA NA 591.6 ± 8.6 384.6 ± 4.6 378.8 ± 5.6 744.8 ± 6.4 127.5 ± 2.3 Ligand 93.5 ± 1.6 114.6 ± 2.7 200.14 ± 3.1 11.89 ± 0.5 25.44 ± 0.8 L-Phenylalanine (L-Phe) N-acetyl tryptophan amide (NATA) N-acetyl tryptophan (NAT) ND ND O -aminobenzoic acid (OAB) a Inhibition constant for free enzyme b Inhibition constant for enzyme–substrate complex With both enzymes, the preference for aromatic amino acids indicated that the aromatic ring offers a better fit at the substrate-binding site Therefore, to analyze the roles of different parts of the inhibitor, a set of good substrate analogs was chosen from the laboratory chemical library Table 3, showing substrate specificity, indicates that tryptophan is a good substrate for both the enzymes Acetylation of the amino group and amidation of the carboxylate group in Trp result in a substrate analog N-acetyl-l-tryptophan amide (NATA) with neutralized charged groups, whereas N-acetyltryptophan (NAT) bears a carboxylate group that is free for interaction The importance of the amino and carboxylate groups could only be assessed with substrate analogs that not have the a-CH in both the groups necessary for hydride transfer OAB is a good substrate analog for this purpose The structures of these inhibitors are shown in Table Inhibition by NATA Assuming that the aromatic rings of the amino acids could play crucial roles in substrate anchoring, NATA is expected to compete with the substrate In reality, NATA between and 135 lm showed uncompetitive rather than competitive inhibition in L1 At higher concentrations up to 540 lm, a mixed inhibition pattern appeared (Fig 3A) The uncompetitive inhibition arose if the inhibitor combined only with the enzyme– substrate complex, i.e when there was no binding site for the inhibitor until a substrate bound to the enzyme [25] The inhibition pattern of Fig 3A has been described as mixed inhibition by some authors and noncompetitive by others [26,27] Alternatively, the term mixed inhibition has been conferred on a special type of noncompetitive inhibition where KI „ KIS This results in double reciprocal plots for different inhibitor concentrations that intersect either above or below the abscissal axis [25] The mixed inhibition pattern appears when the inhibitor combines with both enzyme and enzyme–substrate forms with different affinities and the binding sites are physically separated from the substrate-binding site In principle, if KI>KIS, the inhibition has both noncompetitive and uncompetitive characteristics [25] The presence of a complex inhibition pattern with a distinct uncompeti- FEBS Journal 275 (2008) 2078–2095 ª 2008 The Authors Journal compilation ª 2008 FEBS 2083 Inhibitor-binding sites of L-amino acid oxidase (L1 + NATA) –20 / v (µM/min)–1 x 10–2 C 120 80 40 0 20 40 / [L-phe] M–1 x103 60 (L2 + NATA) 120 –20 –40 80 40 (L2 + NATA) 250 150 C 50 –20 20 40 –1 x 103 / [L-phe] M 60 (Slope replot) D 2.0 1.6 1.2 0.8 0.4 0 –40 / v (µM/min)–1 x 10–2 B Km /Vmax (min) x 10–3 / v (µM/min)–1 x 10–2 A –40 S Mandal and D Bhattacharyya 20 40 60 / [L-phe] M–1 x 103 50 150 250 [NATA] µM Fig Inhibition of L1 and L2 by NATA (A) Double reciprocal plots of inhibition of L1 by lM (e), 105 lM (h), 135 lM (D), 270 lM ( ), 405 lM ( ) and 540 lM ( ) NATA (B) Double reciprocal plots of inhibition of L2 by lM (e), 220 lM (h), 448 lM (D), 896 lM (·) and 1344 lM ( ) NATA (C) The bracketed area of (B) showing competitive inhibition was further analyzed with lM (e), 22 lM (h), 44 lM (D), 88 lM (·), 132 lM ( ), 176 lM ( ) and 220 lM (+) NATA (D) Replot of the slopes of (C) against the concentration of NATA The steady-state kinetic experiments described in Figs 4–7 were carried out at 25 °C in 0.05 M potassium phosphate buffer (pH 6.8) containing 20–100 lM L-Phe as substrate and 30 nM L1 or L2 Arrows in all figures indicate points of intersection tive nature at lower NATA concentrations indicates the involvement of two inhibition mechanisms; we therefore prefer to call it mixed inhibition The intersection of double reciprocal plots at 270–540 lm occurred in the lower left-hand quadrant The kinetic constants for inhibition summarized in Table show that, in this case, KI was 1.5-fold higher than KIS; thus, the mixed inhibition pattern consisted of noncompetitive and uncompetitive components The presence of a mixed inhibition pattern with uncompetitive and noncompetitive characteristics suggests that two NATA molecules bind to L1 at two sites other than the substrate-binding site Inhibition of L2 by NATA followed a mixed inhibition pattern at 0–1344 lm NATA, with an initial competitive inhibition pattern between and 220 lm (Fig 3B) The competitive pattern was examined separately in detail (Fig 3C), focusing on the effect of 2084 inhibitor concentrations on the slopes of double reciprocal plots In principle, a hyperbolic slope replot would indicate that the inhibitor binds to a site on the enzyme other than the substrate-binding site, to show uncompetitive inhibition, as NATA did with L1, and in doing so causes a reduction in Km with an unaltered Vmax Parabolic competitive inhibition is obtained if binding of one inhibitor molecule at the active site facilitates binding of the second inhibitor molecule, so that two molecules of inhibitor contribute to the exclusion of the substrate On the other hand, a linear slope replot would indicate that a single inhibitor molecule binds at the substrate-binding site, resulting in classic competitive inhibition [25] The pattern of competitive inhibition by NATA was verified by employing six inhibitor concentrations ranging from 22 to 220 lm and examining the effect of inhibitor concentration on the slope of the double FEBS Journal 275 (2008) 2078–2095 ª 2008 The Authors Journal compilation ª 2008 FEBS Inhibitor-binding sites of L-amino acid oxidase S Mandal and D Bhattacharyya reciprocal plot Importantly, the slope replot was linear, consistent with the classic competitive inhibition model, where one molecule of NATA interacts with the substrate-binding site (Fig 3D) At higher concentrations of NATA (448–1344 lm), the pattern changed from competitive to mixed inhibition, with KIS being about two-fold higher than KI (Table 2) Mixed inhibition with KIS > KI is known to contain both competitive and noncompetitive components [25] Mixed inhibition of L2 by NATA with initial competitive inhibition suggests that NATA binds at two different sites, of which the substrate-binding site has higher affinity Inhibition by NAT Inhibition of L1 by NAT occurs in two phases: an uncompetitive phase between and 11 lm, followed by mixed inhibition up to 120 lm (Fig 4A) The double reciprocal plots corresponding to inhibitor concentrations producing a mixed inhibition pattern intersect with that of lm in the lower left-hand quadrant, indicating that KI > KIS Inhibition by NAT between and 11 lm showed an uncompetitive profile This was in good correlation with expected lower KIS Increasing the concentration of NAT up to 120 lm favored its binding to both enzyme and enzyme–substrate complex, resulting in a mixed inhibition pattern with uncompetitive and noncompetitive components This pattern of L1 was similar to that with NATA, except that KI and KIS for NAT were 4.6-fold and 4.1-fold lower (Table 2) This indicated that the availability of the carboxyl group facilitates the binding of NAT at uncompetitive and noncompetitive binding sites, but that the aromatic ring and the carboxyl group together are not sufficient for anchoring at the substrate-binding site Inhibition of L2 by NAT also occurred in two phases: it was competitive up to 22 lm, after which there was mixed inhibition up to 88 lm (Fig 4B) The point of intersection of double reciprocal plots ranging from 44 to 66 lm NAT occurred in the upper lefthand quadrant, indicating that KI < KIS Shifting of the initial competitive pattern at 22 lm to a mixed inhibition pattern with an increase in the NAT concentration up to 88 lm results from the binding of the inhibitor to both enzyme and enzyme–substrate complex Therefore, the mixed inhibition appeared as a combination of competitive and uncompetitive patterns Three-fold lower KI and KIS values of NAT as compared to NATA suggest a positive role for the carboxyl group in binding of the inhibitor to the respective sites (Table 2) Fig Inhibition of L1 and L2 by NAT (A) Double reciprocal plots of inhibition of L1 by lM (e), 11 lM (D), 22 lM (s), 44 lM ( ), 88 lM ( ) and 120 lM (+) NAT (B) Double reciprocal plots for inhibition of L2 by lM ( ), 22 lM ( ), 44 lM ( ), 66 lM (·) and 88 lM (:) NAT Inhibition by OAB The inhibitory profiles of substrate analogs studied so far indicate that the aromatic part of the inhibitor, which was sufficient for anchoring at the substratebinding site of L2, leads instead to anchoring at other sites in L1 In both enzymes, the carboxylate group of the inhibitors improved the affinity for the respective sites To determine the importance of the amino group for ligand anchoring, the inhibition kinetics of OAB were investigated The intersection of double reciprocal plots for L1 at different concentrations of OAB occurred at three different points (Fig 5A) The first FEBS Journal 275 (2008) 2078–2095 ª 2008 The Authors Journal compilation ª 2008 FEBS 2085 Inhibitor-binding sites of L-amino acid oxidase S Mandal and D Bhattacharyya point of intersection at lm occurred on the ordinal axis, consistent with the competitive inhibition pattern Upon increase of the OAB concentration to 10 lm, the point of intersection shifted from the ordinal axis to the upper left-hand quadrant, showing mixed inhibition with competitive and noncompetitive components This indicates that another OAB molecule binds at a site physically separated from the substrate-binding site A further increase of the OAB concentration to 25 lm shifted the point of intersection back towards the ordinal axis, showing an unusual inhibition pattern This can happen only when a third OAB molecule binds at another site affecting both the Km and the Vmax Binding of three OAB molecules per enzyme is consistent with the crystal structure of Ca rhodostoma LAAO complexed with OAB [12] The effect of different inhibitor concentrations on the slopes of double reciprocal plots for competitive inhibition was analyzed in a different experiment, using 1–5 lm OAB to confirm the mechanism of competitive inhibition (Fig 5B) Importantly, a replot of the slopes as a function of OAB concentration was linear (Fig 5C), indicating a classic competitive model, in which one inhibitor binds at the substrate-binding site The double reciprocal plot of the inhibition kinetics of L2 at different concentrations of OAB is depicted in Fig 6A The points of intersection of the double reciprocal plots up to 20 lm indicate that OAB inhibited L2 following a mixed inhibition pattern containing an initial competitive component at lm (Fig 6B), similar to the mechanism of inhibition by NATA and NAT The replot of slopes as a function of inhibitor concentration indicated classic competitive inhibition (Fig 6C) At higher concentrations of OAB, between 10 and 20 lm, the point of intersection shifted to the upper left-hand quadrant and remained fixed () A mixed inhibition pattern without further shifting of the intersection point suggests the presence of only two binding sites for OAB in L2 Taken together, these data indicate that the number of inhibitor-binding sites in L2 is two, whereas it is three in L1 Inhibitor cross-competition kinetics The substrate analogs used for predicting modes of inhibition of L1 and L2 showed similar mechanisms, except for OAB The crystallographic structure of the LAAO–OAB complex exhibited three OAB-binding sites at the catalytic funnel Assuming that OAB also binds in the catalytic funnels of L1 and L2, these binding sites were compared with those of NATA and NAT by inhibitor cross-competition kinetics, to determine whether enzyme inhibition in the presence of 2086 Fig Inhibition of L1 by OAB (A) Double reciprocal plots of inhibition by lM ( ), lM ( ), 10 lM ( ), 20 lM (·) and 25 lM ( ) OAB (B) The region indicating competitive inhibition was analyzed further by lM ( ), lM ( ), lM ( ) and lM (·) OAB (C) Replot of the slopes of (B) against the concentration of OAB FEBS Journal 275 (2008) 2078–2095 ª 2008 The Authors Journal compilation ª 2008 FEBS Inhibitor-binding sites of L-amino acid oxidase S Mandal and D Bhattacharyya Fig Inhibition of L2 by OAB (A) Double reciprocal plots of inhibition by lM (e), lM ( ), 10 lM ( ), 15 lM (·) and 20 lM ( ) OAB (B) The region indicating competitive inhibition was analyzed further by lM ( ), lM ( ), lM ( ) and lM (·) OAB (C) Replot of the slopes of (B) against the concentration of OAB two inhibitors arises from simultaneous binding to independent sites or from mutually exclusive binding to a single site or even overlapping sites on the enzyme [28] For this, binary combinations of OAB ⁄ OAB, OAB ⁄ NAT and OAB ⁄ NATA were applied to L1 and L2, considering OAB as inhibitor (I1) and OAB, NAT and NATA as inhibitor (I2) In these experiments, the substrate concentration was held constant throughout, with different sets containing variable concentrations of OAB as I1 Corresponding to each set of [I1] values, OAB, NAT and NATA concentrations were varied as I2 The data were analyzed by plotting the reciprocal of initial velocities as a function of [I1] to visualize the effect on slope while [I2] was varied Variation of OAB concentration in both directions in a binary combination, i.e I1 and I2, was first performed on L1 to validate cross-competition in this system As anticipated, variation of OAB concentration from to 10 lm in one direction, I2, had an effect on the slopes of the reciprocal dependencies obtained from variation of OAB concentration in the second direction, I1, between and lm This was because 10 lm OAB was not sufficient to saturate all three binding sites (Fig 7A) The reciprocal dependencies between the sets of 10 and 20 lm of OAB as I2 eventually became parallel with the one where [I2] = as all the binding sites of OAB became saturated The initial appearance and subsequent disappearance of the slope effect with increasing concentrations of OAB as I2 is consistent with the sequential saturation of the three binding sites The reciprocal plots of the cross-competition between OAB (0–35 lm as I2) and NATA (0– 122.5 lm as I1) showed a complicated intersecting pattern, as NATA could not compete with OAB for all of its binding sites (Fig 7B) At lm OAB, only the substrate-binding site was occupied, leaving other sites free for binding to NATA, and this produced a slope effect The point of intersection between and lm OAB shifted further towards the left with increasing concentrations of OAB between and 25 lm After saturation of three binding sites by OAB at 25 lm, the reciprocal plots for 25 and 35 lm OAB were parallel to each other, indicating mutually exclusive binding of the two inhibitors at two binding sites However, NATA did not compete for binding at the substratebinding site, as there was a slope effect between and 35 lm OAB as I1 A similar intersecting pattern was also observed in the cross-competition between NAT and OAB (Fig 7C), indicating that NATA and NAT bind at the same or overlapping sites where two molecules of OAB also bind, and that binding of OAB at the substrate-binding site is noncompetitive with FEBS Journal 275 (2008) 2078–2095 ª 2008 The Authors Journal compilation ª 2008 FEBS 2087 Inhibitor-binding sites of L-amino acid oxidase S Mandal and D Bhattacharyya Fig Inhibitor cross-competition between OAB and OAB, NATA or NAT in L1 and L2 in the presence of 80 lM L-Phe as substrate (A–C) Cross-competition patterns of L1 between: (A) OAB as indicated and lM ( ), lM ( ), lM ( ), lM (·), 10 lM (h) and 20 lM (D) OAB; (B) NATA as indicated and lM ( ), lM ( ), 25 lM ( ) and 35 lM (·) OAB; and (C) NAT as indicated and lM ( ), lM ( ), 15 lM (·) and 25 lM ( ) OAB (D–F) Cross-competition pattern in L2 between: (D) OAB as indicated and lM ( ), lM (·), 10 lM ( ) and 20 lM ( ) OAB; (E) NATA as indicated and lM ( ), lM ( ), 10 lM ( ) and 20 lM (·) OAB; and (F) NAT as indicated and lM ( ), lM ( ), 10 lM ( ) and 20 lM (·) OAB NATA and NAT In other words, NATA, NAT and OAB compete for binding sites in the catalytic funnel, except at the substrate-binding site 2088 Variation of OAB concentrations in both directions in L2 produced an intersecting pattern at low concentrations of OAB as I2, followed by parallel plots after FEBS Journal 275 (2008) 2078–2095 ª 2008 The Authors Journal compilation ª 2008 FEBS S Mandal and D Bhattacharyya saturation of two of its binding sites (Fig 7D) In OAB and NATA cross-competition experiments, parallel reciprocal plots were observed between and lm OAB as I2, where it is assumed that the two inhibitors compete for the substrate-binding site, based on the KI of OAB (Fig 7E) Widely separated values of KI and KIS for NATA allowed detection of competition at the substrate-binding site Parallel reciprocal plots appeared again between and 20 lm OAB as I1, whereas an intermediate noncompetitive pattern occurred at 10 lm OAB This was the consequence of partial saturation of the second binding site of L2 Competition between OAB and NAT also produced parallel reciprocal plots when the two binding sites were occupied by 20 lm OAB (I1 at and 20 lm, Fig 7F) The intermediate noncompetitive stages indicated saturation of the first binding sites of OAB The cross-competition kinetics in L2 primarily show that the binding sites of OAB, NAT and NATA are either the same or overlapping Discussion Russell’s viper venom contains a number of potent toxins, including PLA2 [29], coagulation factor V and factor X activating proteases [30,31], hyaluronidase [32], hemorrhagins [14–16], and cytotoxins [17] Although LAAOs from several snake venoms are known, there has been no report on LAAO from RVV, except for one that describes the inhibitory property of the ethanolic extract of Tamarindus indica seeds against several toxicological and enzymatic activities of RVV [33] Gel filtration followed by ion exchange chromatography of RVV yielded two fractions of LAAO, termed L1 and L2 (Fig 1) Although SDS ⁄ PAGE could not distinguish between L1 and L2, a difference of kDa was observed in size exclusion HPLC (Fig 1) However, the isoforms showed differences in terms of isoelectric points and amino acid sequences Other characters, such as thermal and pH stability or substrate specificity, were mostly indistinguishable Thus, they may be considered as LAAO isoenzymes according to the definition in [34] The presence of LAAO isoforms in snake venoms is known, but their functional importance has yet to be explored [23,35] The LAAOs characterized so far from snake venoms are dimeric, although some have been reported as monomeric, with a degree of uncertainty [4] The molecular masses of purified L1 and L2 were determined under denaturing and nondenaturing conditions, such as SDS ⁄ PAGE and size exclusion HPLC, where they appeared as monomers of 60–63 kDa The Inhibitor-binding sites of L-amino acid oxidase LAAOs from different sources were reported to contain FAD as a cofactor, except for one from Agkistrodon contortrix laticinctus venom, which contains FMN instead of FAD [36] The UV–visible absorption spectrum and RP-HPLC analysis of the dissociated cofactor from L1 and L2 indicated the presence of FAD (Fig 2) Mass spectral analysis of the cofactor and standard FAD yielded [riboflavin + K]+ with more than 50% abundance The ionization parameters used for ESI MS analysis yielded parent FAD ions with only 20% abundance, and thus the absence of any [FAD + H]+ in the spectrum of the cofactor may be due to low sample concentration and fragmentation during ionization Information from the literature suggests that the majority of LAAOs from different venoms, except for that from Naja hannah, have specificity towards hydrophobic amino acids [4] This preference can be explained on the basis of differences in side chain binding sites within the enzyme [37] Of the hydrophobic amino acids, only five appeared to be good substrates for L1 and L2 All these amino acids have a surface ˚ area above 180 A and a hydrophobicity index close to or above (Table 2) The inability to turn over amino acids with more hydrophobicity and smaller surface area suggests that both enzymes have catalytic sites that require hydrophobic substrates with a minimum ˚ surface area of 180 A to place the a-C within an aver˚ from the flavin N-5 required for an effecage of 3.5 A tive hydride transfer [38] Profiles of the inhibition of L1 and L2 by substrate analogs indicate that the configurations of their catalytic funnels differ from each other A mixed inhibition mechanism in L1 with uncompetitive and noncompetitive components is suggestive of the binding of NATA (neutral form of Trp) at two allosteric sites instead of the substrate-binding site (Fig 3), whereas in L2, it competed with the substrate for the substrate-binding site (Fig 3) Therefore, a difference between the environments of the two substrate-binding sites in terms of ionic character and hydrophobicity is possible The mixed inhibition mechanism with competitive and noncompetitive components indicates that the second site for NATA in L2 is an allosteric binding site that alters the Vmax for NATA (Fig 3) The kinetic data for NATA not support the existence of other allosteric sites in L2, equivalent to the third inhibitor-binding site of L1 The partial N-terminal amino acid sequences of L1 and L2 show 68% and 64% homology with Ca rhodostoma LAAO, suggesting probable structural and functional similarities between them The catalytic site of that enzyme contains FAD as the prosthetic group, FEBS Journal 275 (2008) 2078–2095 ª 2008 The Authors Journal compilation ª 2008 FEBS 2089 Inhibitor-binding sites of L-amino acid oxidase S Mandal and D Bhattacharyya the FAD being deeply buried within the enzyme in a ˚ 25 A long funnel-like entrance The funnel wall also contains electropositive and electronegative residues that guide the amino and carboxylate groups of the substrate amino acid [12] The structures of this region also appeared to be similar in two other snake venom LAAOs from B moojeni and B jararacussu [21] The mixed inhibitions of L1 by NATA and NAT were similar, but the inhibition constant for the latter was lower This 3–4-fold reduced inhibition constant suggests that the free carboxylate group of NAT facilitates its binding as compared to NATA (Fig and Table 3) Moreover, inhibition of L2 by NAT with a lower inhibition constant than that for NATA supports the notion that the binding subsites in both of the isoforms contain hydrophobic and electrostatic surfaces, with the former predominating The entire catalytic funnel of Ca rhodostoma LAAO has three OAB-binding sites The orientations of the three OAB molecules were determined by the electrostatics of the funnel The outermost ligand was positioned at ˚ ˚  10 A within the funnel, the second one at  5.5 A closer to the active site than the outermost OAB, and the third site within the active site nearest to the isoalloxazine ring of FAD The surface closest to the carboxylate groups of those OABs was uniformly electropositive, whereas the surface most proximal to the amino groups was predominantly electronegative [12] A complex inhibition pattern with three points of intersection for double reciprocal plots representing competitive and mixed inhibition mechanisms suggests step-by-step binding of OAB in L1 This took place first at the substrate-binding site, and then at two allosteric sites (Fig 5) The presence of three binding sites in L1 further suggests that the catalytic site, along with the catalytic funnel, may have a degree of similarity to that of Ca rhodostoma LAAO However, the surface of the catalytic funnel wall in L1 is predominantly hydrophobic The inhibitor cross-competition kinetics between OAB, NATA and NAT in L1 supports this, and in addition demonstrates that the two allosteric binding subsites are either the same or overlapping; that is, both of the allosteric sites with predominant hydrophobicity are situated within the catalytic funnel (Fig 8) On the other hand, the inhibition kinetics of OAB suggest the existence of two ligand-binding sites in L2 (Fig 6), one of which is the substrate-binding site Mutually exclusive binding of OAB, NAT and NATA indicates that the binding sites of these inhibitors are the same or overlapping Taken together, all these kinetic findings are indicative of a different catalytic funnel in L2, having one allosteric ligand-binding site of predominant hydrophobicity Binding of OAB in the substrate-binding site was proposed to be similar to binding of the natural substrate in Ca rhodostoma LAAO [12] Recently, a crystal structure of the same enzyme combined with l-Phe has shown similar orientation of the ligand in the catalytic site In the substrate-binding site, the carboxylate group of the ligand was engaged in a salt bridge interaction with the guanidinium group of Arg90 and a hydrogen bond with the hydroxyl group of Tyr372, Fig Proposed catalytic funnels of L1 and L2, showing inhibitor-binding and substrate-binding sites Hydrophobic and electrostatic surfaces have been indicated Intensities of electrostatic surfaces are represented by the number of ‘+’ and ‘)’ signs Intensities of shaded areas indicate strength of hydrophobicity Arrows in L1 indicate rotation of ligand for proper orientation after pivotal anchoring at the electrostatic surface Arrows in L2 indicate rotation of ligand after pivotal anchoring at the hydrophobic surface This hypothesis is based on the ability of the predominantly charged or hydrophobic substrate analogs to bind to the substrate-binding sites of L1 and L2 respectively 2090 FEBS Journal 275 (2008) 2078–2095 ª 2008 The Authors Journal compilation ª 2008 FEBS S Mandal and D Bhattacharyya while the amino group formed a hydrogen bond with the carbonyl oxygen atom of Gly464 The side chain of the ligand participated in hydrophobic interactions with the side chains Ile430, Ile374, and Phe227 [11] On the basis of the crystallographic data and the present findings, we hypothesize that both L1 and L2 have funnel-like catalytic sites but that the distribution and intensity of hydrophobic and charged surfaces are different (Fig 8) The inability of NAT and NATA to bind to the substrate-binding site indicates a predominant electrostatic environment regulating substrate binding in L1 Moreover, the binding of OAB with electropositive, electronegative and hydrophobic groups indicates a pivotal anchoring on the electrostatic residues where the hydrophobic surface directs positioning of the a-C nearest to FAD In contrast to what was found for L1, in L2 NAT and NATA interacted at the substrate-binding site, indicating a predominantly hydrophobic environment regulating the pivotal anchoring at the substrate-binding site, where proper orientation of the amino and carboxylate groups is determined by the respective electrostatic surfaces In summary, RVV contains two LAAO isoforms that are almost indistinguishable in terms of substrate specificity and thermal or pH stability However, the inhibition profiles of the substrate analogs NATA, NAT and OAB in the presence of l-Phe as substrate indicated that the two isoforms were inhibited by different mechanisms A detailed analysis including crosscompetition between the inhibitors has provided insights into the catalytic funnel of the two isoforms It revealed the differences in the environment of catalytic sites in terms of hydrophobic and electrostatic surfaces As both L1 and L2 have significant sequence similarity with the LAAO from Ca rhodostoma, these results have been compared with its crystal structure complexed with OAB The substrate specificity and inhibition data for different substrate analogs indicate that the critical pharmacophore (i.e the minimal structural component required for inhibition) is a hydro˚ phobic aromatic ring of surface area 180–210 A2 provided with carboxylate and amino groups attached to two consecutive carbon atoms This information on inhibitor-binding sites will be helpful in the design of effective suicide substrates for RVV LAAOs Experimental procedures Materials Russell’s viper (Daboia russelli) venom was collected from D Mitra, licensed trophy of Calcutta Snake Park, as desiccated, shining, yellow crystals l-Amino acids, O-dianisidin Inhibitor-binding sites of L-amino acid oxidase dihydrochloride and high molecular mass protein markers (29–207 kDa) were from Sigma-Aldrich (St Louis, MO, USA) Sephadex G-100, Sephadex G-75 and CMSephadex C-50 were from Amersham Biosciences (Uppsala, Sweden) Peroxidase (horseradish, specific activity 280 mg)1) was from Sisco Research Laboratories Ltd (Mumbai, India) Other reagents of analytical grade were purchased locally OAB was a gift from U Halder (Jadavpur University, India) De-ionized water was prepared by passing water through a resin bed (Arium 611DI, Sartorius, Gottingen, Germany) ă Purication of L1 and L2 Venom crystals (21 mg, equivalent to 15 mg of protein) were suspended in mL of 20 mm potassium phosphate buffer (pH 7.2) at 25 °C for 30 min, and the insoluble materials were removed by centrifugation (1000 g, 10 min, °C) [16] The yellowish supernatant was applied to a Sephadex G-100 (117 · 1.2 cm) column pre-equilibrated with the same buffer at °C The flow rate was 16 mLỈh)1, and the fraction size was mL Fractions containing LAAO activity were pooled and loaded onto a CM-Sephadex C-50 (2 · 20 cm) column pre-equilibrated with 20 mm potassium phosphate buffer (pH 7.2) at °C Unabsorbed fractions devoid of LAAO activity were removed by washing with five column volumes of buffer Bound fractions were eluted after application of a linear gradient of 0–0.1 m NaCl in the same buffer (50 + 50 mL) Elution was continued with an additional 100 mL of the final eluent The flow rate was 0.5 mLỈ min)1, and the fraction size was mL Elution was monitored at 280 nm Fractions containing LAAO activity were pooled and concentrated by dialysis against a saturated solution of sucrose in the phosphate buffer The concentrated samples were again dialyzed against 20 mm potassium phosphate buffer (pH 7.2) to remove sucrose, and stored at °C Homogeneity of the samples were verified by 15% SDS ⁄ PAGE and staining with silver nitrate Chromatofocusing Chromatofocusing of the LAAO isoforms was carried out essentially following the method of Amersham Pharmacia Biotech [39] Polybuffer exchanger (PBE 96; Sigma) was packed into a column (0.5 · 14 cm), which was equilibrated with 90 mL of 25 mm Tris ⁄ acetate (pH 8.3) at °C After running mL of elution buffer containing 0.0072 mmolỈ pH unit)1ỈmL)1 PBE 96 (Sigma) (pH 6), pooled fractions from G-100 chromatography with LAAO activity were loaded onto the column The bound proteins were eluted by the elution buffer with a linear flow rate of 12 mLỈh)1, and 1.5 mL fractions were collected The LAAO activity of each fraction was assayed using the coupled assay system, and the pH of each fraction was checked with a pH meter FEBS Journal 275 (2008) 2078–2095 ª 2008 The Authors Journal compilation ª 2008 FEBS 2091 Inhibitor-binding sites of L-amino acid oxidase S Mandal and D Bhattacharyya Native molecular masses of L1 and L2 were determined ˚ from size exclusion HPLC using a Protein-Pak 300 A column (Waters, Milford, MA, USA; fractionation range 10–400 kDa) A Waters 600 HPLC system equipped with a Waters 2487 dual k-absorbance UV–visible detector was used The column was equilibrated with 10 mm potassium phosphate buffer (pH 7.5) containing 100 mm NaCl The flow rate was 0.8 mLỈmin)1, and elution was monitored at 280 nm The column was calibrated with the following molecular mass markers: trypsinogen (24 kDa), carbonic anhydrase (31 kDa), ovalbumin (45 kDa), BSA (67 kDa), and yeast alcohol dehydrogenase (150 kDa) Linear dependency was observed between log molecular mass and Vt (elution time) over of l-amino acid was estimated by horseradish peroxidase in the presence of O-dianisidine The colored product formed was followed continuously by the increase of absorbance at 436 nm (e436 nm = 8.3 mm)1 cm)1) The rate of product formation was linear at least up to 120 s The assay mixture contained 15–100 lm l-Phe (as substrate for LAAO), 350 mU of horseradish peroxidase and 10 lm O-dianisidine (as substrate for peroxidase) diluted with 0.05 m potassium phosphate buffer (pH 6.8) up to mL The temperature of the spectrophotometer cuvette was maintained at 25 °C by a circulating water bath (Polyscience, USA) The reaction was initiated by the addition of 10–20 lL of L1 or L2 (approximately 15–30 nm final concentration) when the reaction rate was optimum However, in a set of experiments where the substrate or inhibitor concentration was varied, the concentration of the enzyme remained constant To verify the substrate specificity, other l-amino acids were used in place of l-Phe under identical assay conditions Identification of the bound cofactor Inhibition kinetics The LAAO fractions from Sephadex G-100 size exclusion chromatography containing mixtures of L1 and L2 were pooled and incubated with 0.1% SDS at 100 °C for 10 The dissociated ligand was separated from the apoenzyme by passage through a Sephadex G-75 (3 · 190 mm) column pre-equilibrated with water Eluted fractions were monitored simultaneously at 280 nm (for proteins) and 450 nm (for the cofactor) Fractions with considerable absorption at 450 nm were scanned between 300 and 600 nm, using water as reference Alternatively, the cofactor was dissociated from the enzyme by heating from 30 to 100 °C over 10 min, and then holding at 100 °C for 10 The denatured protein was removed by centrifugation at 5500 g for The dissociated cofactor was separated from soluble protein by passage through a Millipore Centricon YM 10 filter (Millipore, Billerica, MA, USA) The cofactor was analyzed with a Nova-Pak C18 RP-HPLC column (3.9 · 150 mm, particle size lm), which was equilibrated with 85% solvent A (5 mm ammonium acetate, pH 6.5) and 15% solvent B (100% methanol) at mLỈmin)1 [40] After application of the sample, the column was run with the initial solvent for followed by a linear gradient of 85–25% solvent A (which is equivalent to 15–75% solvent B) over 5–20 Elution of components was followed at 450 nm Reference FAD and eluted cofactor were collected and lyophilized for ESI MS analysis The inhibition kinetics of L1 and L2 were studied in the presence of the substrate analogs NAT, NATA, and OAB NAT (0.0087 m) and NATA (0.0135 m) were dissolved in dimethylsulfoxide and added to the reaction mixture after serial dilution of the stock with buffer The following extinction coefficients were used: NAT, and NATA, e280.8 nm = e279 nm = 5580 m)1 cm)1; 5690 m)1 cm)1 OAB was dried in vacuum desiccators over NaOH pellets to constant weight, weighed, and dissolved in water to prepare a 0.1 m stock During inhibition studies, the inhibitor, the substrate and the coupling enzyme were added to the assay mixture, and the reactions were initiated by the addition of 20 lL of LAAO In control sets, the inhibitors at the concentration applied had no effect on the coupling enzyme (pH 510; Eutech Instruments, Thermo Fisher Scientific, Mumbai, India) Size exclusion HPLC Enzyme assay l-Amino acid oxidase activity was followed by a coupled assay [22] Hydrogen peroxide generated during the turn- 2092 Data analysis Previous reports on the reaction mechanisms of LAAOs from different sources indicated that this enzyme follows Michaelis–Menten kinetics [1,41] The kinetic constants of L1 and L2 with l-amino acid substrates were derived by fitting the initial rate of reaction (v) to the double reciprocal form of the Michaelis–Menten equation (Eqn 1): Km ẳ ỵ v VẵS V 1ị where V, Km and [S] are maximum velocity, Michaelis– Menten constant, and substrate concentration, respectively The mechanism of inhibition was analyzed by a double reciprocal plot of the inhibition kinetics data at different FEBS Journal 275 (2008) 2078–2095 ª 2008 The Authors Journal compilation ª 2008 FEBS S Mandal and D Bhattacharyya Inhibitor-binding sites of L-amino acid oxidase inhibitor concentrations [I], using Eqns (2) and (3) for competitive and mixed inhibition respectively [25,42]:   1 Km ½IŠ ẳ ỵ 2ị 1ỵ v V KI S V ẳ v   ẵI Km 1ỵ ỵ KI V S   ẵI ỵ KIS V 3ị where KI is the inhibition constant for inhibitor binding to the free enzyme, and KIS is the inhibition constant for inhibitor binding to the enzyme–substrate complex The slopes of double reciprocal plots for competitive inhibition were further analyzed by plotting them as a function of [I] to determine the nature of competitive inhibition (linear, parabolic, and hyperbolic) The inhibitor cross-competition pattern was analyzed graphically by using Eqn (4), which is a linear function of ⁄ v versus [I1], as described in [28]:     1 ½I2 ẵI2 ẳ ỵ 1ỵ 1ỵ ẵI1 ð4Þ v v0 Ki2 v0 Ki1 aKi2 where v0 is the initial rate in the absence of inhibitor, Ki1 and Ki2 are the inhibition constants for I1 and I2, respectively, and a is the constant defining the interaction between the two inhibitors Changes in [I2] will have a slope effect if a is close to unity, but will be ineffective if it is infinitely large Therefore, simultaneous binding of two inhibitors to the enzyme will yield reciprocal plots intercepting to the left of the ⁄ v axis On the other hand, reciprocal plots for mutually excluding binding of two inhibitors will be a set of parallel lines [28,43] All the data presented here are means or means ± SD of three independent repeats, and were processed using Microsoft excel Other methods Optical measurements, enzyme assays and spectral scans were done with an Analytic Jena Specord 200 recording spectrophotometer Protein concentrations were determined after Lowry [44], with BSA as reference SDS ⁄ PAGE gels were stained with a Gel Code Glycoprotein staining kit (Pierce, Rockford, IL, USA) for glycoprotein analysis ESI MS (Micromass, Rockford, IL, USA) analysis of cofactor was carried out after dissolving in water Parameters used for ionization were as follows: capillary voltage 3082 V; sample cone voltage 44 V; extraction cone voltage V; desolvation temperature 130 °C; and source temperature 80 °C N-terminal amino acid sequencing was carried out essentially after [17], using an Applied Biosystem (Foster City, CA, USA) automated protein sequencer (model Procise491) Briefly, approximately 100 pmol of L1 and L2 bands from 10% SDS ⁄ PAGE were electrotransfered on an Immobilon PSQ (Millipore) membrane before application to the sequencer The transfer buffer was 10 mm Caps (pH 11) containing 10% methanol Acknowledgements We thank Dr Anil Ghosh for amino acid sequencing and Mr Kalyan Sarkar for MS We also thank Dr Basudeb Acharya for language correction of the manuscript S Mandal was supported by a CSIR-NET fellowship (New Delhi) References Massey V & Curti B (1967) On the reaction mechanism of Crotalus adamanteus L-amino acid oxidase J Biol Chem 242, 1259–1264 Porter DJT & Bright HJ (1980) Interpretation of the pH dependence of flavin reduction in the L-amino acid oxidase reaction J Biol Chem 242, 2969–2975 Curti B, Ronchi S & Simonetta MP (1992) D- and L-amino acid oxidases In Chemistry and Biochemistry of Flavoenzymes, Vol (Muller F, ed.), pp 69–94 CRC Press, Boca Raton, FL Du XY & Clemetson KJ (2002) Snake venom L-amino acid oxidases Toxicon 40, 659–665 Calderon J, Olvera L, Martinez LM & Davila G (1997) A Neurospora crassa mutant altered in regulation of L-amino acid oxidase Microbiology 143, 1969–1974 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