Reaction of Vascular Adhesion Protein-1 (VAP-1) with Primary Amines

Human vascular adhesion protein-1 (VAP-1) is an endothelial copper-dependent amine oxidase involved in the recruitment and extravasation of leukocytes at sites of inflammation. VAP-1 is an important therapeutic target for several pathological conditions. We expressed soluble VAP-1 in HEK293 EBNA1 cells at levels suitable for detailed mechanistic studies with model substrates. Using the model substrate benzylamine, we analyzed the steady-state kinetic parameters of VAP-1 as a function of solution pH. We found two macroscopic pKa values that defined a bell-shaped plot of turnover number kcat,app as a function of pH, representing ionizable groups in the enzyme-substrate complex. The dependence of (kcat/Km)app on pH revealed a single pKa value (∼9) that we assigned to ionization of the amine group in free benzylamine substrate. A kinetic isotope effect (KIE) of 6 to 7.6 on (kcat/Km)app over the pH range of 6 to 10 was observed with d2-benzylamine. Over the same pH range, the KIE on kcat was found to be close to unity. The unusual KIE values on (kcat/Km)app were rationalized using a mechanistic scheme that includes the possibility of multiple isotopically sensitive steps. We also report the analysis of quantitative structure-activity relationships (QSAR) using para-substituted protiated and deuterated phenylethylamines. With phenylethylamines we observed a large KIE on kcat,app (8.01 ± 0.28 with phenylethylamine), indicating that C–H bond breakage is limiting for 2,4,5-trihydroxyphenylalanine quinone reduction. Poor correlations were observed between steady-state rate constants and QSAR parameters. We show the importance of combining KIE, QSAR, and structural studies to gain insight into the complexity of the VAP-1 steady-state mechanism.

group of copper-dependent enzymes that catalyze the oxidative deamination of primary amines to the corresponding aldehydes with concomitant release of hydrogen peroxide and ammonia. (R is a phenyl moiety in case of benzylamine and a benzyl moiety in case of phenylethylamine.) From the viewpoint of structure and mechanism, copper-dependent amine oxidases have been researched extensively for several decades, but relatively little is known about their physiological function. Human vascular adhesion protein-1 (VAP-1) is a dimeric membrane bound and circulating protein that is encoded by the AOC3 gene and is expressed mainly in endothelial cells of blood vessels, adipocytes, and smooth muscle cells (1)(2)(3). VAP-1 is a protein comprising two structural domains: an adhesive domain that targets leukocytes for transmigration and an amine oxidase domain (2,4). At sites of inflammation on the endothelial cell surface, VAP-1 is involved in the recruitment and extravasation of lymphocytes and neutrophils. VAP-1 activity generates important signaling compounds such as hydrogen peroxide (5,6). SSAO activity is also involved in glucose transport in adipose cells (1). As a consequence of the generation of products such as hydrogen peroxide, formaldehyde, and methylglyoxal, which are cytotoxic at high concentrations, the activity of VAP-1 has been linked to cellular damage, including atherosclerosis and vascular complications in diabetes (7,8). Ischemia-reperfusion injury in mice is attenuated through the use of VAP-1-specific inhibitors, and this is confirmed by genetic deletion of VAP-1 in mice (9). The activation of human lung VAP-1 by human plasma is greater if the plasma originates from diabetic patients or following heart myocardial infarction (10).
The physiological substrates for SSAOs are not clearly defined, but the soluble amines methylamine and/or aminoacetone could be among the naturally occurring substrates for SSAOs (11). The majority of studies with copper-dependent amine oxidases have been conducted with the nonphysiological substrate benzylamine, as enzymatic activity with this substrate can be monitored spectroscopically with ease. Efforts to find new substrates for VAP-1 have led to interesting candidates for further study. For example, 1,12-diaminododecane, an aliphatic diamine, is a VAP-1 substrate, and kinetic studies indicate high selectivity compared with other aliphatic (poly)amines (12). For example, the K m with 1,12-diaminododecane is ϳ50-fold lower than with the model substrate benzylamine (12). Bovine VAP has been shown to bind, but not oxidize, lysine and soluble elastin in vitro in the presence of H 2 O 2 and thereby inhibit the catalytic turnover of benzylamine (13). Recently, a binding partner for VAP-1 on the surface of lymphocytes has been identified from phage libraries displaying decapeptides (14). Siglec-10 (sialic acid-binding Ig-like lectin-10), shown to bind specifically to recombinant VAP-1, is the first leukocyte ligand identified for the enzyme (14). Binding results in H 2 O 2 production, suggesting that Siglec-10 acts as a substrate. It was proposed that binding of Siglec-10 occurs via covalent linkage of a free NH 2 group of an arginine side chain to the 2,4,5-trihydroxyphenylalanine quinone (topaquinone (TPQ)) center leading to subsequent oxidation. However, this would be highly unusual in relation to the accepted mechanisms for copper-dependent amine oxidases.
Thus far, the majority of studies have focused on in vivo properties and biomedical investigations of the role of VAP-1 under several pathological conditions (15). Unlike flavin-dependent oxidases, this enzyme uses the unusual active site quinone cofactor TPQ, which is formed autocatalytically. This post-translational modification requires the type-2 copper cofactor and molecular oxygen. The TPQ cofactor facilitates substrate oxidation through the formation of a covalent substrate-cofactor Schiff base intermediate (Scheme 1) in a mechanism that is common to all CAOs. Potentially, VAP-1 is an important target in age-related inflammatory diseases, and it may serve as a marker for chronic liver disease (16). It is therefore surprising that detailed kinetic, mechanistic, and structural studies of VAP-1 are not advanced, as these would provide crucial information to guide rational design of small molecule inhibitors. Here, we explore the mechanism of VAP-1 with respect to amine oxidase activity using model substrates that have been used with other members of the CAO family of enzymes. We report kinetic studies that incorporate analysis of the effects of altering solution pH, isotope effects, and quantitative structure-activity relationships (QSAR) using benzylamine and p-substituted phenylethylamines as substrates. Our study establishes kinetic complexity in the reductive half-reaction of VAP-1 with benzylamine as substrate, consistent with the proposed chemical mechanism. We rationalize the observed isotope effects with benzylamine using a sequential kinetic mechanism, showing that a sequential kinetic mechanism can account for the observed isotope effects on k cat,app , K m.app , and (k cat /K m ) app . Structural models of VAP-1 with the model substrates benzylamine and phenylethylamine bound provide an explanation for the poor correlations we observed in QSAR experiments with p-substituted phenylethylamines. We show the importance of combining kinetic isotope effects (KIE), QSAR, and structural studies when using steady-state kinetic analysis to gain insight into enzyme mechanism.

EXPERIMENTAL PROCEDURES
Materials-Potassium dihydrogen phosphate, dipotassium hydrogen phosphate, 4-aminoantipyrine, 3,5-dichloro-2-hydroxybenzenesulfonic acid, bis-Tris, ␤-phenylethylamine, p-hydroxyphenylethylamine, p-nitrophenylethylamine, and SCHEME 1. A simplified reaction scheme for members of the copper amine oxidase family, indicating the chemical identities of proposed catalytic intermediates. The scheme presented here does not necessarily represent the catalytic mechanism of VAP-1, as detailed spectroscopic studies of VAP-1 have not yet confirmed the presence of the intermediates shown in the catalytic cycle. VAP-1 is represented by E. benzylamine were obtained from Sigma. p-Amino-, p-methyl-, p-fluoro-, and p-chlorophenylethylamine were from Acros Organics. p-Bromophenylethylamine was from Fluorochem and p-methoxyphenylethylamine from Apollo Scientific Ltd. Perdeuterated phenylethylamine (C 6 D 5 CD 2 CD 2 NH 2 HCl, 99.6%) and horseradish peroxidase were from Sigma. Di-deuterated p-substituted phenylethylamines and d 2 -benzylamine were synthesized previously as described by Hothi et al. (17,18). Monoclonal mouse anti-VAP-1 antibody was obtained from Bender Medsystems.
Expression and Purification of VAP-1-The intronless gene that encodes the soluble truncated, form of VAP-1 (sVAP-1; residues 29 -763) was codon-optimized for expression in human cells and synthesized by Qiagen. The gene was synthesized so that it is flanked at the 5Ј-end by a NheI restriction site and a BamHI restriction site at the 3Ј-end, thereby enabling directional subcloning into a modified pCep-Pu vector with an alternative multiple cloning site in which the hygromycin marker was replaced with a puromycin marker (19). This vector contains an N-terminal signal sequence that is derived from a human extracellular glycoprotein (osteonectin), residues 1-19 followed by the sequence Leu-Ala-Ser, which allows extracellular expression of sVAP-1 (20,21).
HEK293 EBNA1 cells were transfected with 2 g of pCep-Pu-sVAP using the transfection reagent Lipofectamine TM (Invitrogen) according to the manufacturer's instructions. The established HEK293 cells were transferred from the original 6-well plate to a fresh 25 ml culture flask after ϳ2.5 weeks of selection. After reaching cell confluence, the medium was replaced with fresh serum-free medium. Cells were cultured for 7 days followed by harvesting of conditioned medium and replacement with fresh medium.
The collected medium, typically 600 ml, was centrifuged and sterilized by passage through a 0.45-m filter (Millipore). CuSO 4 was added to the medium to a final concentration of 0.1 mM, and the medium was incubated at 4°C for at least 6 h (or on ice overnight). The medium containing sVAP-1 was concentrated to 50 ml using an Amicon stirred ultrafiltration cell fitted with a 30-kDa cut-off filter and subsequently concentrated further to 5 ml using a Vivaspin 20 centrifugal concentrator (Sartorius-Stedim). sVAP-1 was purified by size exclusion chromatography using a HiLoad 26/60 Superdex 200 preparative grade column, pre-equilibrated with 20 mM potassium P i buffer, pH 7.6, and 150 mM NaCl at 4°C. The same buffer was used throughout the purification procedure, and sVAP-1-containing fractions were pooled, concentrated, and stored at Ϫ80°C until further use. Purified sVAP-1 was loaded on a 10% polyacrylamide gel for Coomassie Brilliant Blue staining or Western blotting. For Western blot analysis, SDS-PAGE was followed by transfer to a nitrocellulose membrane (Invitrogen). The antimouse WesternBreeze kit from Invitrogen was used according to the manufacturer's protocol. Monoclonal mouse anti-VAP-1 antibody was used to probe sVAP-1.
TPQ Quantification-The amount of TPQ cofactor in sVAP-1 was analyzed by titrating the enzyme with phenylhydrazine, which reacts stoichiometrically with the cofactor (22). A 25 M phenylhydrazine solution was freshly prepared, and 4 l of this solution was added to 0.5 ml of 0.65 mg/ml sVAP-1 (determined using the Pierce BCA protein assay with bovine serum albumin as reference) in 20 mM potassium P i buffer, pH 7.6, and 150 mM NaCl. Optical spectra were recorded frequently to monitor the increase in absorbance at 435 nm until there were no more changes in absorbance (5-15 min). The stepwise addition of phenylhydrazine was repeated until no more spectral changes were observed.
Qualitative Enzyme Activity Measurements-A typical qualitative activity assay used before and during the purification process involved monitoring the oxidation of benzylamine to benzaldehyde. This reaction can be monitored conveniently at 250 nm. The activity assay was performed in a 1-ml reaction volume containing 20 mM potassium P i , pH 7.6, 150 mM NaCl, and 5 mM benzylamine at 37°C for 5 min.
Steady-state Kinetic Assays, pH Dependence, and Kinetic Isotope Effect with Benzylamine-pH-dependent steady-state kinetic studies with benzylamine and d 2 -benzylamine as substrate were performed by following the formation of benzaldehyde at 250 nm (⑀ 250 ϭ 13,800 M Ϫ1 cm Ϫ1 ). Reactions were performed in 180 mM bis-Tris, pH 5.9 -9.2, or in 180 mM sodium bicarbonate buffer, pH 9.2-10.1, with varying concentrations of substrate plus 7.4 nM sVAP-1 (based on TPQ content) at 37°C for 5 min. The kinetic data obtained at pH values of 6.7 and below were fitted with the unmodified hyperbolic Michaelis-Menten equation due the relatively high values for substrate inhibition at lower pH and the difficulty of obtaining reliable data at high benzylamine concentrations. For human and bovine VAP-1 it has been shown that ionic strength influences catalytic turnover, and consequently alternative equations to fit the steady-state data have been proposed (23). For the sake of simplicity, we fitted data displaying substrate inhibition with Equation 1 or Equation 2 (at pH Ͼ 7.7). In these equations i is the initial rate of reaction, V max represents the maximum rate of reaction, S represents substrate concentration, K m represents the Michaelis-Menten constant, K i represents the constant for substrate inhibition, and b is a factor that determines the extent of inhibition. Each data point was obtained from an average of 2-4 measurements.
The pH dependence data were fitted using Equations 3 and 4. For a single kinetically influential ionization, data were fitted using Equation 3, where EH and E are the limiting catalytic efficiencies for the protonated and deprotonated forms of the ionizable groups.
In those cases where two kinetically influential ionizations were observed, the data were fitted using Equation 4, where v m is the pH-independent theoretical maximum rate, and ␣ and ␤ are factors that determine residual activity of the fully protonated and deprotonated enzyme-substrate complex, respectively.
QSARs and KIEs with p-Substituted Phenylethylamines-The effect of para-substitution and deuteration of phenylethylamine on the apparent kinetic parameters of sVAP-1 was investigated by coupling the production of hydrogen peroxide to a chromogenic horseradish peroxidase (HRP) assay. Amine oxidase activity and steady-state kinetic parameters were determined by coupling the production of H 2 O 2 by sVAP-1 to a horseradish peroxidase-mediated oxidation of 4-aminoantipyrine and 3,5-dichloro-2-hydroxybenzenesulfonic acid. This results in the formation of a pink-to-purple-colored product, which can be measured at 515 nm (⑀ 515 ϭ 26,000 M Ϫ1 cm Ϫ1 ) (24). All experiments were performed at 37°C and in 180 mM bis-Tris, pH 7.5. The reaction mixture contained 180 mM bis-Tris, pH 7.5, 0.1 mM 4-aminoantipyrine, 1 mM 3,5-dichloro-2hydroxybenzenesulfonic acid, 3.75 units of horseradish peroxidase, and 11-23 nM sVAP-1 (based on TPQ content).
Electron Paramagnetic Resonance Spectroscopy-Continuous wave EPR spectra were recorded at X-band (ϳ9.4 GHz) using a Bruker ELEXSYS E500/E580 EPR spectrometer (Bruker GmbH, Rheinstetten, Germany). Temperature was controlled using an Oxford Instruments ESR900 helium flow cryostat coupled to an ITC503 controller from the same manufacturer. The temperature was maintained at 20 Ϯ 0.1 K. The microwave power was 0.5 milliwatt, the modulation frequency 100 KHz, and the modulation amplitude 5 G. The g values given were calculated using the software package supplied with the instrument. EPR sample tubes were 4 mm outside diameter in Suprasil quartz supplied by Wilmad. The sample volume was typically 250 l. Samples containing purified oxidized sVAP-1 were frozen in tubes in liquid nitrogen prior to EPR analysis.
Energy Minimization of Structural Models-Structures of the enzyme-substrate imine (Schiff base) complexes for the two substrates, phenylethylamine and benzylamine, were modeled by superimposing the TPQ aromatic ring of the modeled substrate-TPQ imine complex onto the 2-hydrazinopyridinebound TPQ (Protein Data Bank code: 2C11) complex. The atom types and the corresponding force constants to describe the substrate-bound cofactor were assigned by analogy with similar chemical moieties. The equilibrium values for the bonds, angles, and dihedral angles were obtained by optimizing the substrate-bound TPQ using the B3LYP/6 -31G level of theory. The partial atomic charges were computed by RED (25) in conjunction with RESP implemented in AMBER9 (26). The monomeric structure of VAP-1 was then fully solvated in a rectangular water box using the TIP3P model with 8 Å between the edge of the box and the protein. Four Na ϩ ions were added to neutralize the net charge of the system. Minimizations were carried out using the AMBER ff03 force field in AMBER9. The substrate-bound TPQ and protein were first minimized for 200 steps by steepest descent algorithm followed by 1000 steps by conjugate gradient algorithm.

RESULTS AND DISCUSSION
Expression, Purification, and Biochemical Characterization of sVAP-1-For the expression of sVAP-1, part of the gene encoding residues 29 -763 was codon-optimized and synthesized for expression in human cells. The gene was subcloned into a pCep-Pu vector containing the sequence for extracellular expression from human osteonectin (20). Soluble VAP-1 is highly expressed in HEK293 EBNA1 cells and typically represents 50 -90% of the total amount of protein in the medium (Fig. 1). The calculated mass of sVAP-1 is 81,571.9 g/mol, which does not correspond to the apparent mass derived from SDS-PAGE analysis. However, it has been proposed that glycosylation of sVAP is likely responsible for this discrepancy (27). The expressed sVAP-1 was found to be virtually inactive (0.3 unit/ ml) because of the low concentration of copper in the medium. The addition of 0.1 mM Cu II resulted in a ϳ23-fold increase in activity (6.7 units/ml) following overnight incubation. The yield of sVAP-1 after purification using size exclusion chromatography ranged from 10 to 59 mg/liter of culture medium (n ϭ 6 purifications). The expression of sVAP-1 was markedly higher than what had been reported by others (27). To calculate the amount of active sVAP-1, the concentration of TPQ cofactor was determined by titrating the enzyme against phenylhydrazine, which forms a stable covalent adduct with the cofactor. The formation of this adduct can be monitored spectroscopically ( Fig. 2A). Upon the addition of phenylhydrazine, a linear increase in absorbance at 435 nm was observed, which reached a maximum value at 14.72 M phenylhydrazine ( Fig. 2A, inset). Longer incubation times resulted in a further slow increase in absorbance, attributed to the known instability of phenylhydrazine in neutral to alkaline buffered solutions (28). The concentration of total protein was determined using the bicinchoninic Blue. The scanned gel shows purified sVAP-1, which runs at ϳ100 kDa. B, Western blot using a VAP-1 monoclonal antibody confirmed that the band identified using Coomassie Brilliant Blue staining was indeed the correct protein. sVAP-1 is indicated by the arrow; M is the prestained protein molecular weight marker from Stratagene.
acid assay (79.3 M). This indicated that ϳ19% of the purified sVAP-1 had assembled the TPQ cofactor, suggesting that a significant proportion (ϳ81%) of the sVAP-1 lacked Cu II or that the concentration of oxygen in solution was limiting during the autocatalytic formation of TPQ. However, the addition of copper to the growth media, prolonged incubation with copper after purification, or purging with molecular oxygen did not increase the apparent TPQ content. We inferred therefore that either Cu II was replaced by another metal in a large fraction of the purified VAP-1 proteins or that the TPQ cofactor was not able to react with phenylhydrazine, perhaps because of the altered structure of the active site.
We performed EPR analysis with 85 M purified sVAP-1 to confirm the presence of type-2 copper and investigated whether other metals might occupy the copper binding site. The EPR spectrum (Fig. 3) is axial and clearly shows the typical type-2 copper peaks attributed to the coordination of a Cu II ion by three histidine residues and a water molecule, with g ʈ ϭ 2.27, g Ќ ϭ 2.06, and A ʈ ϭ 163 G. The slight tailing of the peaks suggests that a second Cu II environment may be present in a small percentage (less than 10%) of the molecules. This second environment may result from the presence of a hydroxide ion instead of the water molecule close to the Cu II , arising from the ionization of water at pH 7.6, or it might be the result of alternate conformations of the TPQ cofactor which can swing between "on-copper" and "off-copper" states. Crystal structures of VAP-1 show that the TPQ cofactor can shuttle between a conformation in which it interacts directly with Cu II (oncopper), rendering the cofactor unavailable for catalysis, and a catalytically active conformation in which the TPQ O5 is positioned near the active site catalytic base Asp-386 (off-copper) (29 -31). The possibility that Cu II is present as a mixture of 63 Cu II and 65 Cu II has been considered; however, the differences in Cu II hyperfine coupling between 63 Cu II and 65 Cu II are small compared with the EPR line width, and so the contributions from the two isotopes superimpose almost exactly. No other paramagnetic metals were detected. We quantified the type-2 copper in sVAP-1 based on a 100 M CuEDTA standard and found that Ͼ95% of the purified sVAP-1 enzyme contains type-2 copper. This is in line with our previous observation that prolonged incubation with Cu II ions does not improve the TPQ:enzyme ratio of purified sVAP-1. Other workers have also reported low TPQ content in related amine oxidases such as pig plasma amine oxidase and Hansenula polymorpha amine oxidase (32,33). Our observations suggest that the TPQ cofactor may be largely in the on-copper conformation, which renders the cofactor unavailable for catalysis.
We analyzed the spectral characteristics of purified sVAP-1 by recording the optical spectra of oxidized and benzylaminereduced enzyme under anaerobic conditions. The concentrated purified enzyme has a "peach pink" color, which is typical of TPQ-containing enzymes and is indicative of a broad absorbance band around 480 nm (34). The oxidized minus reduced difference spectrum of sVAP-1 shows an absorbance maximum at 476 nm with an extinction coefficient of 2.9 mM Ϫ1 cm Ϫ1 (Fig.  2B). This is comparable with what has been reported for other TPQ-containing amine oxidases (22,34).   Fig. 4A depicts the pH dependence of k cat,app with benzylamine as substrate. The graph shows a bellshaped curve with a maximum theoretical k cat,app value of 2.99 Ϯ 0.11 s Ϫ1 and two pK a values of 7.0 Ϯ 0.2 and 10.0 Ϯ 0.4, respectively. The macroscopic pK a values derived from this plot represent pK a values of ionizable groups of the enzyme-substrate complex. The lower pK a,1 value is attributed to one or more groups that need to be deprotonated for activity, whereas the alkaline pK a2 represents one or more groups that need to be protonated for optimal enzyme catalysis.

Dependence of Apparent k cat and D (k cat ) on Solution pH with Benzylamine Substrate-
The k cat,app values obtained with d 2 -benzylamine were similar to the values obtained with protiated benzylamine, yielding KIEs close to unity (Fig. 4C). This means that proton abstraction from the ␣-carbon of benzylamine (going from the substrate Schiff base to the product Schiff base in Fig. 1) is not rate-limiting in steady-state catalysis. It should be noted that the experiments were conducted in air-saturated buffers. Upon saturation of the buffers with molecular oxygen, which corresponds to a ϳ5-fold increase in concentration of molecular oxygen in solution, the k cat,app increased by a factor of only 1.11 Ϯ 0.04 at pH 7.2. This suggests that reoxidation of the aminoquinol form of sVAP-1 by oxygen does not significantly limit the observed rate of catalysis under these conditions. In addition, the steady-state kinetic parameters at pH 7.5 with benzylamine as substrate were determined in H 2 O and D 2 O to investigate whether a solvent isotope effect could be observed. In this case, we calculated a solvent isotope effect of 1.46 Ϯ 0.05, suggesting that one or more steps in the kinetic mechanism involving the attack of a water molecule (e.g. imine hydrolysis; see Scheme 1) or proton transfer are partly rate-limiting.
Dependence of Apparent k cat /K m and D (k cat /K m ) on Solution pH with Benzylamine Substrate-The pH dependence of (k cat / K m ) app of sVAP-1 with benzylamine displays an S-shaped curve with a pK a value of 9.0 Ϯ 0.1 (Fig. 4B). This indicates that an ionizable group on the free substrate or enzyme must be deprotonated for efficient catalysis. The determined pK a value is similar to the pK a for the primary amine of benzylamine, which is 9.3 (35). We infer that the pK a value is likely attributable to the primary amine of benzylamine, as upon deprotonation of the amine the nitrogen lone pair becomes available for nucleophilic attack on the C5 of the TPQ cofactor (34). Binding of substrate to the enzyme can significantly perturb pK a values, which can lead to different observed pK a values in the k cat versus the k cat /K m data. For example, the ionization pK a of the primary amine group on the substrate can be perturbed by ϳ2-3 orders of magnitude (36). Therefore, the lower observed pK a value of 7.0 from the k cat dependence data may represent the perturbed pK a of the primary amine group on benzylamine. This perturbation is essential, as lowering of the pK a results in a net increase of deprotonated amine in the active site with a lone pair then available for nucleophilic attack and allows the enzyme to function with unprotonated substrate at physiologically relevant pH values (36).
A KIE of 6 -7.6 is observed across the pH profile upon deuteration of the substrate (Fig. 4D); the only exception is at pH  4); B, a single pK a value (9.0 Ϯ 0.1) was calculated from the S-shaped (k cat /K m ) app versus the pH dependence curve. The pH dependence of kinetic isotope effects on k cat,app (C) and (k cat /K m ) app (D) was calculated by dividing the steady-state kinetic parameter with protiated benzylamine by the respective steady-state kinetic parameter with d 2 -benzylamine. In both cases the KIE appears to be pH-independent; the KIE on k cat is close to unity, and the KIE on (k cat /K m ) app is ϳ6 -7.6. D indicates that this is the isotope effect on k cat : D (k cat ) ϭ k cat of the protiated substrate divided by k cat of the deuterated substrate.
5.9, where the KIE is lower (4.1 Ϯ 0.7). The relatively large K m,app at pH 5.9 complicates the data collection at higher substrate concentrations. This K m,app was calculated using the unmodified Michaelis-Menten equation, and as VAP-1 exhibits substrate inhibition with benzylamine, this might therefore contribute to the lower calculated KIE (23). The equal drop in catalytic efficiency over the pH range for protiated and deuterated benzylamine likely relates to the lack of significant change in the pK a value for the ionization of the amine. This agrees with previous findings that dideuteration of the ␣-carbon of benzylamine results in a low secondary isotope effect on benzylamine basicity (⌬pK a of 0.032 Ϯ 0.001) (37).
The KIE on (k cat /K m ) app is largely due to an increase in K m,app , with the deuterated substrate as compared with the protiated benzylamine. A comparable observation has been made with aromatic amine dehydrogenase (AADH) from Alcaligenes faecalis, where, albeit to a lesser extent, isotopic substitution causes an increase in the apparent K m value (38). Both k cat,app and K m,app are complex functions of the kinetic mechanism of VAP-1, which are built up of the individual rate and binding constants. When observing a large D (k cat /K m ) app but a D (k cat ) app close to unity, the first possible explanation to be explored would be the presence of a binding isotope effect (BIE) as a result of the altered bond vibrational environment caused by the isotopic substitution (39). However, BIE values are typically close to unity, suggesting that the observed KIE is not solely attributable to a hypothetical BIE (39). 4 In an attempt to shed light on the possible mechanistic cause of this unusual isotope effect, we performed algebraic and numerical analyses on the rate equations and equations describing the steady-state kinetic parameters for the proposed simplified kinetic mechanism of VAP-1 (Scheme 2). In the supplemental material, we provide details of the equations and the modeling used to analyze the effects on measured kinetic parameters following isotopic substitution. To simplify the analysis, in Scheme 2 product release and the oxidative halfreaction were grouped into one step (k 4 ), as the oxidative halfreaction does not involve benzylamine. We derived equations for D (k cat /K m ) app and D (k cat,app ) assuming an isotopically sensitive reversible deprotonation of the ␣-carbon of the substrate Schiff base (k 3 /k Ϫ3 ) (see supplemental material). This option could only explain the observed KIEs if we assumed a large value for the reverse step (k Ϫ2 ) preceding the isotopically sen-sitive step, such that k Ϫ2 Ͼ Ͼ k 2 , k Ϫ2 is in the same order of magnitude as k 3 , and k 4 is rate-limiting. Although this assumption is not impossible, we also set out to find a solution that would be more plausible in terms of the reverse rate constants. A similar isotope effect on (k cat /K m ) app has been observed in other enzyme systems (40). In this case attempts were made to rationalize such effects by incorporating additional branched pathways in the kinetic mechanism. The mechanism proposed to be responsible for the observed large KIE on (k cat /K m ) app of cytochrome P-450IIE1 includes a loop that leads the oxenoid complex that is about to enter an isotopically sensitive step back to the enzyme-substrate complex (40). However, the addition of such a loop to a typical CAO catalytic mechanism cannot be related to a realistic chemical event.
In principle, any mechanistic step that involves a labeled molecule may exhibit an isotope effect (41). Inspection of the proposed reductive half-reaction of CAOs reveals several steps that are likely to display an isotope effect. Substrate reactivity depends on the protonation state of the primary amine, and the pK a value for this group is slightly increased upon isotopic substitution at the ␣-carbon (42). The nucleophilic attack of the substrate amine lone pair on the C5 carbonyl of TPQ, which leads to the formation of a carbinolamine intermediate, may be sensitive to isotopic substitution. Including this isotopically sensitive nucleophilic attack in our calculations improves the outcome of the numerical analyses (Table S1). It appears that the observed KIE on (k cat /K m ) app with benzylamine as substrate can be best explained by a weighted average of the intrinsic isotope effects on the individual isotopically sensitive steps (41) Equation 5.
Quantitative Structure-Activity Relationships with para-Substituted Phenylethylamines-As proton abstraction from the substrate Schiff base with benzylamine is not rate-limiting, QSAR studies with this substrate will not yield informative data. Therefore, we decided to use p-substituted phenylethylamines for further analyses, considering that different substrates may exhibit different kinetic behavior. QSAR studies are based on the Hammett methodology, which allows for the derivation of correlations between kinetic parameters and substituent constants (43,44). The constants that are used in these correlations are based on electronic ( p , which is composed of field and inductive effects (F) and resonance effects (R)), hydrophobic (), and steric effects (E s ). F describes the transmittance of electrical influence of a substituent to the reactive group via electrostatic interactions through space (field) and the electrical influence, which is primarily transmitted by polarization of the -bonds (inductive). R reflects the ability of a group to donate electrons to or withdraw electrons from the conjugated system of -electrons.
In many QSAR studies, substituted benzylamines are the first choice as substrates because of the relatively straightforward activity assay and varying degrees of electronegativity. With benzylamine as substrate, no KIE on k cat,app was observed, so it was decided to pursue studies with para-substituted phenylethylamines and their deuterated counterparts to investigate structure-activity correlations. The prerequisite in the case of steady-state kinetic analysis is that there is a significant KIE on k cat,app so that C-H and C-D bond breaking is (at least partly) SCHEME 2. Kinetic scheme for the reductive half-reaction of VAP-1 used as model for algebraic and numerical calculations of kinetic isotope effects in the VAP-1 catalytic mechanism with benzylamine as substrate. In this scheme, E represents the enzyme, S the substrate, and P the product; ES is the Michaelis complex, ESS is the substrate Schiff base intermediate, and EPS is the product Schiff base. This kinetic scheme is based on the proposed chemical scheme for VAP-1 by analogy with other copper amine oxidases (see Scheme 1).
rate-limiting. The reactivity of sVAP-1 with phenylethylamine was monitored spectroscopically by coupling the production of H 2 O 2 to a chromogenic assay catalyzed by HRP. Attempts were made to use this assay to study the pH dependence of k cat,app and (k cat /K m ) app , but at pH Ͼ 8 the assay proved to be unreliable. Benzylamine was used as a control substrate at different pH values to validate the activities obtained from this coupled assay by comparing them with the rate of benzaldehyde formation at 250 nm. This revealed that the sVAP-1 activity measured using the HRP-linked assay underestimated the rate of reaction at pH Ͼ 8 when compared with reactions employing benzylamine as substrate (monitored at 250 nm). Increasing the amount of HRP did not increase the observed rate of reaction, but increasing the concentration of 4-aminoantipyrine and 3,5dichloro-2-hydroxybenzenesulfonic acid resulted in a increase of the observed rate. To minimize complications that arise from the use of the HRP assay at elevated pH values, we performed all QSAR experiments at pH 7.5.
The experimentally determined steady-state kinetic parameters for sVAP-1 with para-substituted protiated and deuterated phenylethylamines are presented in Table 1. The large KIEs on k cat,app indicate that abstraction of the C␣-proton is rate-limiting. Very poor correlations of enzyme reactivity are observed with field/inductive and resonance effects (Fig. 5, B and C). The resonance value of a substituent increases with the ability to withdraw electrons from the conjugated aromatic system. Based on the chemistry of the rate-limiting step, electronwithdrawing substituents are expected to increase the rate of reaction. This is in accordance with the k cat,app of sVAP-1 with NO 2 -phenylethylamine, which is 6-fold higher than with OHphenylethylamine. The resonance values for Cl-, Br-, and CH 3phenylethylamine are similar, and similar rates would be expected, but only with the halogenated substrates was this found to be true. A 3-fold difference in k cat,app was observed between CH 3 -phenylethylamine and Cl-phenylethylamine, which confirms the weak correlation pattern. In Fig. 5B no correlation between k cat,app and the field/inductive effect is observed. When NO 2 -phenylethylamine is excluded from the electronic effects plot (Fig. 5A), a positive slope is observed, which implies a positive correlation between k cat,app and electronic properties of the substituents. Taking into account the positive correlation, NO 2 -phenylethylamine is expected to cause a larger increase in activity. It may be that in this case the rate of C-H/D bond breakage is not (fully) rate-limiting. As with the R-effect, the overall correlation in this case is weak. Furthermore, no correlation was observed with hydrophobic or steric effects.
The weak correlations can be partially explained by the additional methylene group between the C␣ and the aromatic ring in phenylethylamine as compared with benzylamine. This increase in distance between the substituent and the C␣ atom attenuates field/inductive effects (45). The size of the active site cavity of VAP-1, in particular Leu-469, has been suggested to limit the size of VAP-1 substrates (46). In an effort to visualize the implications of para-substitution in the enzyme active site, structures of VAP-1 with benzylamine or phenylethylamine bound in the substrate Schiff base form (based on Protein Data Bank code: 2C11 (30)) were modeled and energy-minimized. The modeled structures of VAP-1 in the substrate Schiff base intermediate form show that the presence of phenylethylamine has a potentially large effect on the conformation of the side chain of Leu-469, as it is displaced from its position in the substrate-free form of the enzyme by the aromatic ring of the substrate (Fig. 6). This in turn leaves little room for para-substituents of phenylethylamine. The inferred steric clash and structural reorganization will likely contribute to the poor observed QSAR correlations.
Unlike with benzylamine, with phenylethylamines as the substrate we observed large KIEs on the turnover number k cat,app . This indicates that the observed rate reflects the rate of TPQ reduction through C␣-H/D bond cleavage. Model structures of VAP-1 bound to benzylamine and phenylethylamine show that the overall orientation of the benzylamine Schiff base intermediate, and in particular the geometry of the methylene moiety, which is involved in H-transfer to the catalytic base Asp-386, is different compared with the phenylethylamine Schiff base (Fig. 6) (31). With benzylamine the pro-S and pro-R protons are pointing more toward the O1 of Asp-386 (2.55 and 2.78 Å, respectively) than toward the O2 of Asp-386 (2.78 and 2.85 Å, respectively). Contrastingly, with phenylethylamine as substrate the pro-S and pro-R protons are pointing slightly more toward the O2 of Asp-386 (2.55 and 3.14 Å, respectively) than toward the O1 of Asp-386 (2.78 and 3.39 Å, respectively). The model structures show that Asp-386 has no clear preference for abstraction of the pro-S or pro-R proton on benzylamine, which is in accord with a previous report on VAP-1 stereochemistry (47). The orientation of the C␣ protons on the phenylethylamine Schiff base model combined with the hydro-

determined with para-substituted protiated and deuterated phenylethylamines
Initial rates of substrate oxidation were measured by coupling hydrogen peroxide production to a horseradish peroxidase-catalysed chromogenic assay. The assays were performed at 37°C in 180 mM bis-Tris, pH 7.5, 0.1 mM 4-aminoantipyrine, 1 mM 3,5-dichloro-2-hydroxybenzenesulfonic acid, 3.75 units of horseradish peroxidase, and 11-23 nM sVAP-1 (based on TPQ content). The steady-state parameters for protiated and deuterated benzylamine are indicated by the preceding superscripts H and D, respectively. ND, not determined.  (48). In VAP-1 a similar stabilizing effect between the Tyr-473 hydroxyl group and the O2 of Asp-386 could contribute to a decrease in the rate of reduction, causing proton abstraction to become rate-limiting with phenylethylamine as substrate.

para-Substituent
Conclusions-We were able to drive high level production of VAP-1 in HEK293 EBNA1 cells, which has allowed us to explore steady-state kinetic behavior, incorporating analysis of isotope effects and QSAR relationships. The apparent substoichiometric TPQ cofactor content and relatively weak spectral properties complicate the transient kinetic analyses, which is why the steady-state studies presented here add significantly to our knowledge of the kinetic mechanism of human VAP-1. Our studies emphasize the importance of using a broad approach to the analysis of kinetic mechanisms by coupling QSAR and isotope effect data with models of active site structure. Recent studies on tryptophan tryptophylquinone-dependent AADH also show that caution should be taken when using QSAR studies to analyze the chemistry of enzyme mechanism. The use of isotope effects in the case of AADH reveals that p-substituted benzylamines are poor reactivity probes, as proton abstraction is not fully rate-limiting (18). Our isotope effect studies with benzylamine suggest a complex kinetic mechanism for VAP-1, consistent with mechanistic schemes for the reductive half-reactions of related CAOs. Furthermore, we show how small changes in the molecular structure of the substrate, such as one FIGURE 5. Quantitative structure-reactivity relationships of sVAP-1 k cat,app values with para-substituted phenylethylamines and their deuterated counterparts. The k cat,app values obtained, as presented in Table 1, have been plotted against the electronic, field/inductive, and resonance parameters for the para-substituted phenylethylamines. A, plot of log k cat,app versus the electronic effect of para-substituted protiated phenylethylamines (F) and deuterated phenylethylamines (E). B, same as A but with reactivity versus field/inductive effects. C, same as A but with reactivity versus resonance effect. FIGURE 6. Energy-minimized model structures of VAP-1 with benzylamine or phenylethylamine bound. Structures of the enzyme-substrate imine (Schiff base) complexes for the two model substrates benzylamine (BE, light gray) and phenylethylamine (PEA, dark gray) were modeled by superimposing the TPQ aromatic ring of the modeled substrate-TPQ imine complex onto the 2-hydrazinopyridine-bound TPQ (Protein Data Bank code: 2C11) complex. Minimizations were carried out using the AMBER ff03 force field in AMBER9. Indicated distances are in Å. With benzylamine the pro-S and pro-R protons are pointing more toward the O1 of Asp-386 (2.55 and 2.78 Å, respectively) than toward the O2 of Asp-386 (2.78 and 2.85 Å, respectively). With phenylethylamine as substrate, the pro-S and pro-R protons are pointing slightly more toward the O2 of Asp-386 (2.55 and 3.14 Å, respectively) than toward the O1 of Asp-386 (2.78 and 3.39 Å, respectively). extra methylene moiety in phenylethylamine compared with benzylamine, result in different kinetic effects on VAP-1 mechanism. This in turn indicates that kinetic studies with model substrates may not always reflect the enzyme kinetics of an enzyme with its physiological substrate. Therefore, care should be taken with the development and analysis of novel substrates and inhibitors for VAP-1. A detailed knowledge of the kinetic mechanism of VAP-1 will aid in the development of new substrates and inhibitors as potential treatments for several pathological conditions (12,46,49).