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Chem. Res. Toxicol. 2006, 19, 102-110

Inhibition of Human Mitochondrial Aldehyde Dehydrogenase by 4-Hydroxynon-2-enal and 4-Oxonon-2-enal† Jonathan A. Doorn,‡ Thomas D. Hurley,§ and Dennis R. Petersen*,| DiVision of Medicinal and Natural Products Chemistry, College of Pharmacy, The UniVersity of Iowa, Iowa City, Iowa 52242, Department of Biochemistry and Molecular Biology, UniVersity of Indiana School of Medicine, Indianapolis, Indiana 46202, and Department of Pharmaceutical Sciences, School of Pharmacy, The UniVersity of Colorado Health Sciences Center, DenVer, Colorado 80262 ReceiVed July 8, 2005

Previous studies found the lipid peroxidation product 4-hydroxynon-2-enal (4HNE) to be both a substrate and an inhibitor of mitochondrial aldehyde dehydrogenase (ALDH2). Inhibition of the enzyme by 4HNE was demonstrated kinetically to be reversible at low micromolar aldehyde but may involve covalent modification at higher concentrations. Structurally analogous to 4HNE is the lipid peroxidation product 4-oxonon-2-enal (4ONE), which is more reactive than 4HNE toward protein nucleophiles. The goal of this work was to determine whether 4ONE is a substrate or inhibitor of human ALDH2 (hALDH2) and elucidate the mechanism of enzyme inhibition by 4HNE and 4ONE. Both 4ONE and its glutathione conjugate were found to be substrates for the enzyme in the presence of NAD. At low concentrations of 4ONE (e10 µM), hALDH2 catalyzed the oxidation of 4ONE to 4-oxonon-2-enoic acid (4ONEA) with a maximal yield of 5.2 mol 4ONEA produced per mol of enzyme (monomer). However, subsequent analysis of hALDH2 activity toward propionaldehyde revealed that both 4ONE and the oxidation product, 4ONEA, were potent, irreversible inhibitors of the enzyme. In contrast, inhibition of hALDH2 by a high concentration of 4HNE (i.e., 50 µM) was primarily reversible. The reactivity of 4ONEA toward glutathione was measured and found to be comparable to that of 4HNE, indicating that the 4ONE-oxidation product is a reactive electrophile. hALDH2/NAD was incubated with 4HNE, 4ONE, and 4ONEA, and mass spectral analysis of tryptic peptides revealed covalent modification of an hALDH2 active site peptide by both 4ONE and 4ONEA. These data demonstrate that hALDH2 catalyzes the oxidation of 4ONE to 4ONEA; however, the product 4ONEA is a reactive electrophile. Furthermore, both 4ONE and 4ONEA are potent, irreversible inhibitors of the enzyme. Introduction Cellular oxidative stress can initiate lipid peroxidation, yielding reactive aldehydes capable of covalently modifying proteins and DNA (1). 4-Hydroxynon-2-enal (4HNE)1 was found to be a major, cytotoxic product of the oxidative decomposition of lipids (1, 2). 4HNE is an R,β-unsaturated aldehyde that was demonstrated to be reactive toward the protein nucleophiles Cys, His, and Lys via Michael addition (3-5). Pathways for cellular metabolism of 4HNE have been elucidated and include enzyme-mediated oxidation, reduction, and glutathione (GSH) conjugation (6). Mitochondrial aldehyde dehydrogenase (ALDH2; EC 1.2.1.3) was found to catalyze oxidation of the lipid aldehyde to the nonelectrophilic 4-hydroxynon-2-enoic acid (4HNA) in a NAD-dependent manner (7, 8). While 4HNE was a substrate for the enzyme, it was also † Presented in part at the Enzymology and Molecular Biology of Carbonyl Metabolism 12th International Meeting, Burlington, VT, July 7-11, 2004. * To whom correspondence should be addressed. Tel: 303-315-6159. Fax: 303-315-0274. E-mail: [email protected]. ‡ The University of Iowa. § University of Indiana School of Medicine. | The University of Colorado Health Sciences Center. 1 Abbreviations: 4HNA, 4-hydroxynon-2-enoic acid; 4HNE, 4-hydroxynon-2-enal; 4ONE, 4-oxonon-2-enal; 4ONEA, 4-oxonon-2-enoic acid; ALDH2, mitochondrial aldehyde dehydrogenase; DMP, Dess-Martin periodinane; GS-4ONE, glutathione-4ONE conjugate; GS-4ONEA, glutathione-4ONEA conjugate; GSH, glutathione; hALDH2, human recombinant mitochondrial aldehyde dehydrogenase; RT, retention time; TIC, total ion chromatogram.

found to be a potent mixed type inhibitor (i.e., Ki ) 0.5 µM) of ALDH2-mediated acetaldehyde oxidation (9). Although the enzyme contains an active site Cys nucleophile (i.e., Cys 302 in the human enzyme; 10, 11) involved in catalysis that could be modified by the lipid aldehyde, later work demonstrated kinetically that inhibition of ALDH2 by 4HNE was reversible at low micromolar concentrations (12). However, it is conceivable that at higher concentrations, the active site thiol(s) is covalently modified by the lipid aldehyde. 4-Oxonon-2-enal (4ONE) was demonstrated to be a reactive product of lipid peroxidation (3, 13-15), and recent studies have identified reductases that biotransform the lipid aldehyde to a less electrophilic form (16, 17). 4ONE is structurally analogous to 4HNE but more reactive toward thiols and amines (3). Because of this property, it is possible that 4ONE is a suicide substrate for metabolic enzymes that interact with 4HNE, thus increasing the biological half-life of 4HNE. The present study was undertaken to accomplish the following: first, to determine whether 4ONE is a substrate for ALDH2 and ascertain the reactivity of the product(s) and second, to ascertain whether 4ONE is an inhibitor of the enzyme and elucidate the mechanism of inactivation. Recombinant human ALDH2 (hALDH2) was obtained and incubated with 4ONE and NAD, and the activity was measured toward the lipid aldehyde and its GSH conjugate. The product, 4-oxonon-2-enoic acid (4ONEA), was synthesized, and its reactivity toward GSH was determined. Inhibition of hALDH2 with 4HNE, 4ONE, and

10.1021/tx0501839 CCC: $33.50 © 2006 American Chemical Society Published on Web 12/23/2005

Aldehyde Dehydrogenase Inhibition by Lipids

4ONEA was assessed, and liquid chromatography-tandem mass spectrometry (LC-MS/MS) was used to identify lipid adducts on tryptic peptides. The enzyme activity toward 4ONE and its GSH conjugate (GS-4ONE) was demonstrated; however, both 4ONE and the oxidative product, 4ONEA, were found to be potent, irreversible inhibitors that yield covalent modifications on hALDH2.

Materials and Methods Materials. GSH, NAD, and silver nitrate were purchased from Sigma (St. Louis, MO). Dess-Martin periodinane (DMP) was obtained from Aldrich (Milwaukee, WI). Desalting columns were purchased from Pierce (Rockford, IL). Trypsin (sequencing grade, modified) was obtained from Promega (Madison, WI). HPLC grade water and acetonitrile were purchased from Fisher (Fairlawn, NJ). All other chemicals (e.g., solvents and buffers) were of the highest possible grade. Synthesis of 4HNE, 4ONE, and 4ONEA. 4HNE was synthesized as the dimethyl acetal and liberated from the acetal via acid hydrolysis or as the free aldehyde, according to procedures described elsewhere (18, 19). 4ONE was prepared by oxidizing 4HNE with DMP (3). 4ONEA was synthesized via oxidation of 4ONE using silver oxide (20) and confirmed via UV/vis, GC/MS, and NMR analysis. NMR spectra were recorded at 300 MHz on a Bruker Avance-300 spectrometer with δ values in ppm. 1H NMR (CDCl3): δ 7.18-7.13 (d, 1H, J ) 15.8 Hz), 6.66-6.72 (d, 1H, J ) 16.1 Hz), 2.64-2.69 (t, 2H, J ) 7.17 Hz), 1.29-1.37 (m, 6H), 0.92-0.95 (t, 3H). 13C NMR (CD3SOCD3): δ 201.05 (CdO), 167.29 (COOH), 139.91 (C3, CdC), 131.99 (C2, CdC), 31.33 (C7, CH2), 23.49 (C6, CH2), 22.59 (C8, CH2) and 14.49 (CH3). C5 (CH2), predicted to be about δ 41, was masked by the DMSO signal at δ 39.3-41.0. Preparation of hALDH2. hALDH2 was prepared as described previously (21-24) and stored in 50% glycerol. Before use, hALDH2 was dialyzed against a 4000-fold excess of 50 mM sodium phosphate buffer, pH 7.4, for at least 4 h to remove glycerol. The protein concentration was measured using the BCA assay (Pierce). Preparation of GSH Conjugates. GS-4ONE was prepared by incubation of 1 mM 4ONE with 1.5 mM GSH in 50 mM sodium phosphate buffer, pH 7.4, for 5 min at room temperature (23 ( 1 °C). Conjugation of 4ONE with GSH was confirmed by an absence of a sharp peak at 228 nm, corresponding to loss of the 4ONE enone system. The GSH conjugate of 4ONEA (GS-4ONEA) was prepared in a similar manner, except an incubation time of 30 min was used, and conjugation of 4ONEA with GSH was confirmed by an absence of a sharp peak at 222 nm, corresponding to loss of the 4ONEA enone system. Enzyme Activity. All assays were performed in 50 mM sodium phosphate buffer, pH 7.4, at 37 °C. The activity was assessed by monitoring the production of NADH at 340 nm ( ) 6220 M-1 cm-1) using a Molecular Devices SpectraMax 190 plate reader (Sunnyvale, CA). 4ONE was used at concentrations ranging from 5 to 100 µM with 1 mM NAD. Total NADH produced was obtained via 340 nm absorbance at t ) 1800 s. GS-4ONE was used at 100 and 400 µM with 1 mM NAD, and kinetic parameters were calculated using progress curves (25). Determination of 4ONEA Reactivity. GSH adduction was carried out with the [GSH] g 10 [4ONEA] in 50 mM sodium phosphate buffer. A change in [4ONEA] was monitored by a decrease in absorbance at 222 nm. The rate constant (k) was determined from three independent experiments. Linear regresssion and statistical analysis were performed using GraphPad Prism 3.02 (GraphPad Software, San Diego, CA). LC/ESI/MS Confirmation of hALDH2 Activity toward GS4ONE. Five to eight micrograms of hALDH2 was incubated with 1 mM NAD and 100 µM GS-4ONE for 1 h at 37 °C. LC/ESI/MS analysis was accomplished using an Agilent 1100 Series Capillary LC and MSD Ion Trap SL (Agilent Technologies, Palo Alto, CA). One microliter of product was injected via an autosampler, and

Chem. Res. Toxicol., Vol. 19, No. 1, 2006 103 separation was accomplished with gradient elution using a Phenomenex C18 column (150 mm × 1 mm i.d.; 300 Å) (Torrance, CA) at a flow rate of 50 µL/min. The solvents used were 0.2% formic acid in water (A) and 0.2% formic acid in acetonitrile (B) with gradient conditions as follows: 5% B at 0 min, 5% B at 5 min, 20% B at 15 min, 40% B at 35 min, 70% B at 40 min, 90% B at 42 min and held for 2 min, and 5% B at 45 min. Mass spectrometric detection and analysis were accomplished using positive ion mode with a capillary voltage of 3.5 kV. The nebulizer pressure was set at 20 psi, and dry gas flow was set at 8 L/min with the temperature of the dry gas set to 350 °C. The scanning range for all analyses was 50-700 m/z. MS/MS analysis was accomplished using the Auto MSn feature with the fragmentation amplitude set to 1.5 V. Standards of 100 µM GS-4ONE and 100 µM GS-4ONEA were prepared, incubated for 1 h at 37 °C, and analyzed in the same manner. Inhibition of hALDH2 by 4HNE, 4ONE, and 4ONEA. Five micrograms of hALDH2 was incubated with 1.0 mM NAD and 50 µM 4HNE, 4ONE, or 4ONEA in 50 mM sodium phosphate buffer, pH 7.4, at 37 °C. At time points, 5 µL of sample was removed and added to 195 µL of buffered solution (i.e., 50 mM sodium phosphate, pH 7.4) containing 1 mM propionaldehyde and 1 mM NAD at 37 °C. The activity was measured via an increase in absorbance at 340 nm as described above. Inhibition of hALDH2 by 4ONE and 4ONEA was further characterized as follows. Five micrograms of hALDH2 was incubated with 0.5 mM NAD and [4ONE] ) 0.50, 1.0, 2.0, and 5.0 µM or [4ONEA] ) 1.0, 2.0, 5.0, 10, 15, and 20 µM. At time points, 180 µL of buffered solution (i.e., 50 mM sodium phosphate, pH 7.4) containing 1 mM propionaldehyde and 1 mM NAD was added to each 20 µL sample. The activity was measured as described above. Analysis of data to determine kinetic parameters for 4ONEA-mediated inhibition was performed using GraphPad Prism 3.02 (GraphPad Software). Preparation of Modified hALDH2 for LC-MS/MS Analysis. A 2.5 µM concentration of hALDH2 was incubated with 1 mM NAD and 100 µM 4HNE, 4ONE, or 4ONEA for 30 min at 37 °C in 50 mM sodium phosphate buffer, pH 7.4. After the 30 min incubation, an aliquot was withdrawn and assayed with a 40-fold excess of buffered 1 mM propionaldehyde and 1 mM NAD as described above to verify enzyme inactivation. Inhibitor (i.e., 4HNE, 4ONE, or 4ONEA) was removed via either washing with a 2-fold excess of CH2Cl2 or using a Pierce desalting column (according to instructions specified by the manufacturer). 1,4-Dithiothreitol and acetonitrile were added to final concentrations of 0.5 mM and 10% (v/v), respectively. The protein was denatured by incubation at 70 °C for 3 min. Proteolytic digestion was accomplished via treatment with trypsin (Promega) at a final concentration of 1:20-1:40 (µg trypsin:µg protein) for at least 4 h at 37 °C. LC-MS/MS Analysis of Modified hALDH2. Tryptic peptides of hALDH2 (8 µL injection volume) were analyzed by LC-MS/ MS using an Agilent 1100 Series LC/ESI-MSD Trap equipped with a Phenomenex Jupiter C18 column (1 mm × 150 mm, 300 Å). The mobile phase consisted of (A) 0.2% formic acid in water and (B) 0.2% formic acid in acetonitrile with a flow rate of 50 µL/min and gradient conditions as follows: 5% B at 0 min, 5% B at 5 min, 70% B at 30 min, 90% B at 33 min and held for 10 min, and 5% B at 43 min and held for 2 min. Mass spectrometric detection and analysis were accomplished using the positive ion mode with a capillary voltage of 3.5 kV. The nebulizer pressure was set at 20 psi, and dry gas flow was set at 8 L/min with the temperature of the dry gas set to 350 °C. The scanning range for all analyses was 400-1800 m/z. Peptides within the mass range of 500-1500 Da were subject to MS/MS analysis. Spectral deconvolution and data analysis were accomplished using the LC-MSD trap software (Bruker Daltonik).

Results hALDH2 Activity toward 4ONE. hALDH2 was found to have activity toward 4ONE in the presence of NAD. Enzyme

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Figure 1. Activity of hALDH2 toward 4ONE, evident from enzymemediated reduction of NAD to NADH in the presence of substrate.

Figure 3. LC/MS confirmation of hALDH2/NAD activity toward GS4ONE. (A) Product of incubation of GS-4ONE with hALDH2/NAD for 1 h at 37 °C. (B) Chromatogram for the standard GS-4ONE. (C) Chromatogram for GS-4ONEA standard.

Figure 2. Correlation between product yield and [hALDH2]. At 10 µM 4ONE, the total amount of substrate oxidized varied linearly with [hALDH2], with slope ) 5.2 ((0.55) mol 4ONE/mol hALDH2 monomer. Enzyme-catalyzed oxidation of 4ONE was determined via measurement of NAD reduction to NADH.

was incubated with 5-100 µM 4ONE and 1 mM NAD, and the activity was assessed via an increase in absorbance at 340 nm, corresponding to NADH production. As shown in Figure 1, NADH production was evident for all concentrations of substrate used; however, as demonstrated by this figure, there was a negative correlation between substrate concentration and product yield (i.e., NADH). A linear correlation was evident between hALDH2 concentration and maximal product yield when a low 4ONE concentration was used (i.e., 10 µM). As shown in Figure 2, a graph of pmol of hALDH2 (i.e., catalytic monomer) vs pmol of NADH yielded a slope of 5.2 ((0.55). Such a result indicates that one catalytic monomer of hALDH2 will produce 5.2 NADH molecules when 4ONE is used as substrate. The Presumed 4ONE Oxidation Product, 4ONEA, Is an Electrophile. Oxidation of 4ONE to 4ONEA is presumed to result in a product containing an R,β-unsaturated ketone, predicted to be a Michael acceptor. This speculation was confirmed by experiments demonstrating 4ONEA to be reactive toward GSH, as indicated by loss of absorbance at 222 nm when the two chemicals were coincubated. Pseudo-first-order kinetics were observed under the conditions used (i.e., [GSH] in g10fold excess over [4ONEA]), indicating that the conjugation reaction is second-order as expected. The bimolecular rate constant (k) for modification of GSH by 4ONEA was determined to be 1.65 ((0.146) M-1 s-1. hALDH2 Catalyzes Oxidation of GS-4ONE. To evaluate the possibility that GS-4ONE is also a substrate for hALDH2, the GSH conjugate of 4ONE was prepared and incubated with 1 mM NAD and enzyme. GS-4ONE was found to be a substrate for hALDH2, as evident by a linear increase in absorbance at 340 nm corresponding to enzyme-catalyzed reduction of cofactor to NADH. Vmax and kcat (i.e., for the monomer) were determined to be 44.4 ((1.45) nmol/min/mg protein and 2.42 ((0.0803) min-1, respectively. Using progress curves, the KM was

calculated to be 183 ((32.6) nM, yielding a catalytic efficiency of 1.31 (0.240) × 107 M-1 min-1. hALDH2 activity toward GS-4ONE was confirmed via analysis of product using LC-MS/MS. As shown in Figure 3, incubation of enzyme with 100 µM GS-4ONE and 1 mM NAD for 1 h at 37 °C yielded the oxidized GSH conjugate (GS4ONEA). Two peaks at 20.4 and 21.0 min were evident in the total ion chromatogram (TIC) with MH+ at m/z 478.4 (Figure 3A) consistent with the predicted mass for GS-4ONEA. Furthermore, the retention time (RT) of the two peaks was identical to a prepared GS-4ONEA standard (Figure 3C). Residual GS-4ONE (MH+ at m/z 462.4) was observed and eluted with a RT of 19.4 min as a broad peak (Figure 3A). A large peak at 28.3 min was observed in Figure 3A with MH+ at m/z 426 and also in the TIC for the GS-4ONE standard (Figure 3B) but not the GS-4ONEA standard (Figure 3C). MH+ of such a compound is consistent with that for GS-4ONE (-2H2O) and could represent the product of reaction of the GSH free amine with the 4ONE-carbonyl(s). Inhibition of hALDH2 by 4HNE, 4ONE, and 4ONEA. 4HNE, 4ONE, and 4ONEA are electrophiles/Michael acceptors that are predicted to modify protein thiols and amines and, therefore, could irreversibly inhibit hALDH2 by forming covalent adducts. To determine whether 4HNE, 4ONE, and the product of enzyme-catalyzed 4ONE oxidation (i.e., 4ONEA) are irreversible inhibitors of hALDH2, the enzyme was incubated with 50 µM of each lipid and 1 mM NAD. At time points, the enzyme plus inhibitor and cofactor were diluted with a large excess (i.e., 1:40, v:v) of substrate solution composed of 1 mM propionaldehyde and 1 mM NAD. Therefore, decreased enzyme activity following dilution would correspond to irreversible inhibition. As shown in Figure 4, hALDH2 incubated with 50 µM 4HNE lost only e10% activity over the 30 min time course. In contrast, enzyme treated with 50 µM 4ONE or 4ONEA was inhibited >90%. Such results indicate that inhibition of hALDH2 by 4HNE is primarily reversible; however, enzyme inhibition by 4ONE or 4ONEA is primarily irreversible, involving covalent adduction of the protein. As shown in Figure 5A,B, treatment of hALDH2 over a short period of time (i.e., 20 min) with low micromolar 4ONE or 4ONEA results in inhibition of activity. Interestingly, the plot for inactivation of the enzyme by 4ONEA appeared (pseudo)

Aldehyde Dehydrogenase Inhibition by Lipids

Figure 4. Inhibition of hALDH2 in the presence of 1 mM NAD by 50 µM 4HNE, 4ONE, or 4ONEA. At time points, hALDH2/NAD/ inhibitor was diluted 1:40 with substrate solution ([propionaldehyde]final ) 1 mM, [NAD] final ) 1 mM), and the activity was measured.

first-order, and this was confirmed by log transformation of the data (Figure 5C). Slopes of the log-linear plot, i.e., pseudofirst-order rate constants (k′), varied linearly with [4ONEA], and saturation was not observed over the concentration range used (Figure 5D). A bimolecular rate constant for hALDH2 inactivation of 185 ((13.0) M-1 s-1 was calculated from the plot of k′ vs [4ONEA]. Modification of hALDH2 by 4HNE, 4ONE, and 4ONEA. To identify sites of hALDH2 alkylation, the enzyme was incubated with 50 µM each inhibitor for 60 min, digested, and analyzed via LC-MS/MS. Before proteolytic digestion, hALDH2 treated with 4HNE, 4ONE, or 4ONEA was diluted 1:40 (v:v) with substrate solution of 1 mM propionaldehyde and 1 mM NAD and assayed for activity as before to verify inhibition. Peptides were separated using HPLC and analyzed via an ion trap mass spectrometer as described in the Materials and Methods. Incubation of hALDH2 with 4ONE and 4ONEA but not 4HNE resulted in modification of the tryptic peptide containing the active site thiol (i.e., Cys 302; Table 1). The peptide containing Cys 302 (residues 273-307; calculated MH+ at m/z 3862.7; measured MH+ at m/z 3864.8) was present in the

Chem. Res. Toxicol., Vol. 19, No. 1, 2006 105

chromatograms for the control (Figure 6A) and 4HNE-treated samples (RT ) 20.5 min) but not those for enzyme incubated with 4ONE or 4ONEA. Only when a high concentration of 4HNE (i.e., 500 µM), the active site peptide was modified (Table 1), corresponding to a loss of >90% activity after 1:40 dilution in substrate solution ([propionaldehyde]final ) 1 mM; [NAD]final ) 1 mM). As shown in Table 1 and Figure 6, treatment of hALDH2 with 4ONE or 4ONEA resulted in modification of the active site peptide containing the catalytic Cys 302. Multiply charged ions at m/z 807.8 (5+), 1009.4 (4+), and 1345.7 (3+) of the active site peptide were observed in the chromatogram at 21.6 min (Figure 6B,C), with subsequent deconvolution yielding the singly charged ion at m/z 4034.7 (Figure 6D and Table 1), corresponding to the active site peptide with a 169.9 Da adduct (i.e., 4ONEA). These data demonstrate modification of the tryptic peptide containing Cys 302 by 4ONE and 4ONEA and 4HNE at a higher concentration (i.e., 500 µM). Several other tryptic peptides were found to be modified by 4ONE, 4ONEA, and 4HNE at a higher concentration, as shown in Table 1. Sequence coverage for the hALDH2 protein was typically >80%. The peptides found to contain adducts had free cysteines, and on the basis of reactivity toward thiols, it is likely that these are the target residues. Interestingly, hALDH2 treated with 4ONE resulted in the presence of 4ONEA adducts, based on mass (170 Da). An N-terminal peptide (1-34) was found to have a 4ONEA modification, exclusively, and the active site fragment (273-307) was determined to contain either a 4ONE or a 4ONEA adduct. The 4ONEA adduct could be the result of auto-oxidation of the aldehyde group on the 4ONE modification. On the basis of fragmentation via MS/MS analysis, Cys 302 appears to be the target residue for 4ONE (as a 4ONEA adduct) and 4ONEA modification on the active site tryptic peptide (i.e., residues 273-307), which contains three free thiols (i.e., Cys 301, Cys 302, and Cys 303), as shown in Figure 7. The quadruply charged peak (at m/z 1009.4) of the 35-residue peptide containing the 4ONEA adduct was subjected to fragmentation,

Figure 5. Inhibition of hALDH2/NAD by low micromolar 4ONE and 4ONEA. (A) Time course for inactivation of hALDH2/NAD by 4ONE. (B) Enzyme inhibition by 4ONEA. (C) Log-linear plot for 4ONEA-mediated hALDH2 inactivation, demonstrating pseudo-first-order kinetics. The slope of each curve represents k′ (min-1). (D) Linear correlation of k′ (min-1) vs [4ONEA], with slope ) k (µM-1 min-1) for enzyme inhibition.

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Table 1. hALDH2 Tryptic Peptides Containing 4HNE, 4ONE, or 4ONEA Adducts treatmenta 50 µM 4HNE 500 µM 4HNE 50 µM 4ONE

50 µM 4ONEA

peptideb

RT (min)

modified

unmodified

adductc

439-469 100-127 1-34 156-178 273-307d 1-34 273-307d 273-307d 156-192e/178 1-34 273-307d 156-178

22.5 22.9 19.6 22.7 22.9 20.0/33.0f 20.7/37.5f 35.4f 20.7e/38.3f 20.2/32.7f 21.6/35.4f 23.0/37.5f

3510.4 3315.6 3910.0 2819.2 4019.0 3920.2 4020.6/4018.3 4035.0 2970.2/2834.4 3924.1/3922.2 4034.7/4035.2 2834.9/2835.3

3355.7 3159.7 3754.2 2663.1 3864.8 3754.2 3864.8 3864.8 2663.1 3754.2 3864.8 2663.1

154.7 155.9 155.8 156.1 154.2 166.0 155.8/153.5 170.2 307.1e/171.3 169.9/168.0 169.9/170.4 171.8/172.2

a Tryptic peptides were obtained and analyzed as described in Material and Methods. b Numbering of residues in hALDH2 protein. c Adduct mass deduced from subtracting mass of unmodified from modified peptide. d Peptide containing the active site thiol nucleophile, Cys 302. e Peptide found to have mass corresponding to two 4ONE adducts. f The second RT shown refers to additional analysis performed using the extended HPLC gradient as follows: 5% B at 0 min, 5% B at 5 min, 70% B at 50 min, 90% B at 53 min and held for 10 min, and 5% B at 63 min and held for 2 min.

yielding the following major ions, consistent with an adduct on Cys 302: b2 (412.2), y5 (493.2), b5 (525.3), b6-NH3 (639.3), b6 (656.4), b7-NH3 (726.4), b7 (743.4), y6-H2O (748.3), y6 (766.4), y7-H2O (851.2), y7 (869.3), b9-NH3 (913.1), y8 (997.2), y9-H2O (1036.4), y9 (1054.4), b11 (1175.3), y10 (1182.4), y12NH3 (1426.5), y12 (1443.6), b21-H2O (2311.6), b21 (2329.8), b22NH3 (2426.8), b22 (2443.0), b23-H2O (2572.9), b23 (2589.9), b24NH3 (2719.8), b24 (2737.9), b26 (2979.1), b28 (3164.0), b31 (3644.2), and b34 (3859.3). The y5, y6, and y7 ions, which map the 4ONEA adduct to Cys 302, were found to be prevalent in the MS/MS spectrum, at 17, 16, and 93% relative intensity, respectively (Figure 7). Fragmentation of the 4ONEA adduct resulting from treatment of hALDH2 with 4ONE yielded a similar result (data not shown), thus confirming adduction of Cys 302.

Discussion Previous studies demonstrated 4HNE to be a substrate for rat liver ALDH2, with Km and Vmax values of 14.3 µM and 3.5 nmol min-1 mg protein-1, respectively (7, 8). Not surprisingly, hALDH2 was found to have activity for the structurally analogous 4ONE, differing from 4HNE by a ketone at C4 instead of a hydroxyl group in the present study. However, the kinetic characteristics were remarkably different for each substrate with the enzyme. ALDH2 with 4HNE and NAD exhibited Michealis-Menten kinetics and increased activity with higher [substrate] (7, 8). In contrast, primary kinetic curves of the enzyme with 4ONE and cofactor, measured by a change in absorbance at 340 nm based on enzyme-mediated NAD reduction to NADH, were nonlinear in the absence of substrate and/or cofactor depletion as shown in Figure 1.2 Furthermore, the overall product yield, based on maximal change in absorbance, was inversely proportional to [4ONE] at concentrations >10 µM (Figure 1). Such findings demonstrate 4ONE to be a substrate for hALDH2 and enzymecatalyzed oxidation of the lipid aldehyde; however, 4ONE or the product 4ONEA may be an inhibitor of hALDH2. At low concentrations of the substrate, [4ONE] < 10 µM, the overall product yield had positive correlation with concentration of both substrate and hALDH2. By titrating product yield 2 For 5 µM 4ONE substrate concentration, 1000 pmol of 4ONE was present in the sample well, and ∼800 pmol was enzymatically oxidized as apparent from ∼800 pmol of NADH produced. On the basis of the calculations, the ALDH2 reaction appears to have completed before the substrate was consumed; however, it is possible that the 200 pmol unaccounted for auto-oxidized or reacted with protein or cofactor in the assay; therefore, the enzymatic reaction went to completion.

with [enzyme], it was found that 1 mol of hALDH2 (monomer) could catalyze oxidation of 5.2 ((0.55) mol of 4ONE, deduced from measured total NADH production (Figure 2). On the basis of calculations, consumption of substrate or cofactor was not a limiting factor,2 demonstrating inhibition of the enzyme by 4ONE even at low micromolar concentration. Such a result may indicate the degree of competition between 4ONE as substrate vs inhibitor, as the lipid aldehyde is a reactive electrophile (i.e., Michael acceptor) that could potentially modify the active site Cys 302. An alternative explanation is that approximately one in five 4ONEA exits the active site not through the substrate channel but via the opposite side of the active site where the reduced cofactor resides, thus exposing the catalytic Cys 302 to the electrophilic carbon of the 4ONE oxidation product. Future experiments involving the addition of Mg2+ to the assay would provide insight in regards to this proposal, as Mg2+ stabilizes the position of the nicotinamide ring and could, therefore, reduce frequency of encounters between Cys 302 and 4ONEA (26). Because the acid product of 4ONE oxidation contains an R,βunsaturated ketone capable of being a Michael acceptor, it was incubated with GSH to determine reactivity of this electrophile. 4ONEA was found to react with GSH, as evident by a decrease in the absorbance at 222 nm, representing a GS-4ONEA conjugate lacking the enone system. The rate of the reaction varied with [GSH], and the k was measured to be 1.65 ((0.146) M-1 s-1. As compared to the rate constant for conjugation of GSH with 4ONE (i.e., k ) 145 M-1 s-1; 3), 4ONEA is significantly less reactive toward GSH, that is, by a factor of ∼90. However, the 4ONE oxidation product is still reasonably reactive, especially when compared to the well-studied 4HNE, considered to be a strong biological electrophile (k ) 1.1-1.3 M-1 s-1 for GSH; 1, 3). Therefore, both 4ONE substrate and presumed oxidation product are reactive Michael acceptors that could modify cellular proteins. On the basis of the impressive reactivity toward GSH (3) and high concentrations of the tripeptide in many cell types (e.g., g5 mM), 4ONE generated in vivo may exist predominantly as GS-4ONE. Previous work found that the GSH conjugate of the R,β-unsaturated aldehyde, acrolein, was a substrate for rat liver mitochondrial ALDH (27). Therefore, it was of interest to determine whether hALDH2 has activity toward the GSH conjugate of 4ONE. Results obtained from enzyme kinetic analysis and product confirmation using LC/MS demonstrated GS-4ONE to be a substrate for hALDH2/NAD. The kcat was calculated to be low (i.e., 2.42 min-1) as compared with other substrates, such as aliphatic aldehydes (28, 29); however, the

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Figure 6. (A) TIC for tryptic peptides of control hALDH2. The unmodified active site peptide containing the catalytic Cys 302 (residues 273307) eluted at 20.5 min. (B) Extraction ion chromatogram of hALDH2 incubated with 50 µM 4ONEA and subsequently digested with trypsin for the peak at m/z 1009.4, corresponding to the quadruply charged active site peptide (residues 273-307) with a 4ONEA adduct. (C) Spectrum at 21.7 min with peaks representing the multiply charged active site peptide (residues 273-307) containing a 4ONEA modification, specifically shown are the 3+, 4+, and 5+ ions. (D) The deconvoluted spectrum for the peaks representing the multiply charged active site peptide (residues 273-307) containing a 4ONEA adduct, specifically, the peak at m/z 4034.7.

Figure 7. Active site hALDH2 tryptic peptide containing the catalytic Cys 302, shown in bold as C30 in sequence. b and y fragment ions from MS/MS analysis, as reported in the Results section, locate the 4ONEA adduct to C30 (i.e., Cys 302).

Km was determined to be in the submicromolar range at 183 nM, yielding a reasonable overall catalytic efficiency of 1.31 × 107 M-1 min-1. Interestingly, the Km is significantly lower

than that measured for the GSH conjugate of acrolein (i.e., 198 µM; 27), which may be due to the hALDH2 preference for longer chain aldehydes (28). As noted in the Results section, LC/MS analysis of the reaction product of GS-4ONE with hALDH2/NAD demonstrated enzyme-mediated oxidation of the GSH conjugate to GS4ONEA (MH+ at m/z 478.4; Figure 3). Two major peaks were present in the TIC with RTs similar to those for the GS-4ONEA standard, which was produced via reaction of GSH with 4ONEA. Because 4ONEA is only reactive at C2 toward GSH, the peaks in the TIC most likely represent GS-4ONEA stereoisomers resulting from GSH conjugated with the C2 of 4ONE that is subsequently oxidized. It is conceivable that hALDH2

108 Chem. Res. Toxicol., Vol. 19, No. 1, 2006

has a preference for one stereoisomer over the other; however, this was not determined in the present study. While 4ONE as substrate resulted in inactivation of hALDH2, the enzyme was not inhibited by GS-4ONE at concentrations used (i.e., 100 and 400 µM). Limited turnover of GS-4ONE by hALDH2/NAD was not observed, as shown for 4ONE, and kinetic parameters were readily measurable. However, these results do not demonstrate increased catalytic efficiency of GS4ONE as compared to 4ONE, as observed for aldose reductase/ NADPH (16, 30, 31), which has a GSH binding site resulting in higher affinity for GSH conjugates vs parent aldehydes (32). No evidence was acquired demonstrating the presence of a GSH binding site on hALDH2. Instead, the data obtained most likely reflect the decreased reactivity of the GS-4ONE conjugate as compared to the 4ONE parent compound. GS-4ONE contains two carbonyls but lacks the enone system (i.e., Michael acceptor); therefore, the current findings suggest that inhibition of hALDH2 may be due to thiol, as opposed to amine, modification. Initial experiments with 4ONE as substrate showed inactivation of hALDH2/NAD by 4ONE even at low micromolar concentrations; therefore, work was performed to elucidate the mechanism of enzyme inhibition. Because 4ONEA is an electrophile with reactivity comparable to that of 4HNE, it was used in experiments to determine whether the oxidation product inhibited hALDH2. Incubation of hALDH2/NAD for 30 min with 50 µM 4ONE or 4ONEA resulted in nearly complete inactivation (i.e., >90%) of enzyme activity toward propionaldehyde after dilution in substrate solution. Such results indicate irreversible enzyme inactivation by 4ONE and 4ONEA most likely due to covalent modification of hALDH2 catalytic residues. Previous studies found 4HNE to be a potent, reversible inhibitor of hALDH2 at low micromolar concentrations (9, 12). However, it is conceivable that at high micromolar concentrations, 4HNE could irreversibly inhibit the enzyme. In contrast to the results with 50 µM 4ONE or 4ONEA treatment, hALDH2 incubated for 30 min with an equivalent concentration of 4HNE was inhibited only 90% of enzyme inhibition by 4HNE is reversible even at 50 µM of the lipid aldehyde. Only with 500 µM 4HNE treatment was irreversible inactivation observed along with modification of the active site peptide containing Cys 302 (data not shown). It is conceivable that hALDH2 was adducted by the 30 min incubation with 50 µM 4HNE, but residues modified are therefore not critical for enzyme activity. However, while not affecting activity, adduction of hALDH2 by 4HNE in a cell could potentially have biological significance, e.g., altered protein turnover (33), yielding a change in mitochondrial chemistry. To determine sites of hALDH2 modification by 4ONE and 4ONEA, hALDH2/NAD was treated with each inhibitor and digested with trypsin. The resulting peptides were fractionated using HPLC and analyzed using an ion trap. As shown in Table 1, several sites of modification were identified, most notably, the active site peptide containing the catalytic thiol. Cys 302 serves as a nucleophile in the ALDH2-catalyzed oxidation of aldehydes to acids (10, 11) and, therefore, represents a likely target for 4ONE and 4ONEA, which would result in enzyme inactivation. However, the catalytic thiol is flanked on both sides by Cys residues (i.e., Cys 301 and 303; 33), which could also be modified by the lipids, and a previous study demonstrated the reactivity of Cys 301 toward alkylating agents in the cytosolic enzyme (34).

Doorn et al. Scheme 1

For 4ONE, a potential mechanism for hALDH2 inhibition could involve nucleophilic reaction of Cys 302 with the aldehyde yielding a thiohemiacetal intermediate, followed by Michael addition of Cys 301/303 with C2 of the lipid aldehyde (Scheme 1). Such a mechanism might explain the apparent biphasic kinetics of hALDH2 inactivation by 4ONE. MS/MS fragmentation of the active site peptide indicated modification of Cys 302 by the lipid aldehyde as the 4ONEA adduct (Figure 7). However, the 4ONEA modification could result from oxidation of 4ONE followed by Cys 302 alkylation by the 4ONEA product. Molecular modeling experiments could provide insight on this topic. The mechanism proposed in Scheme 1 would not be plausible for 4ONEA due to the presence of a carboxylate at C1, but it is conceivable that Cys 301, 302, or 303 could react with 4ONEA and/or that all three thiols are valid targets resulting in a mixture of products/adducted species (e.g., 1:1, Cys 301:Cys 302 adducted hALDH2). However, on the basis of two pieces of evidence presented here, Cys 302 appears to be the target of the 4ONE oxidation product. First, the k was determined for inactivation of hALDH2 and calculated to be 185 ((13.0) M-1 s-1, which is >100-fold greater than the rate constant for reaction of GSH with 4ONEA. Such evidence demonstrates that the thiol modified by 4ONEA is “activated” (i.e., polarized to increase reactivity; 36), such as Cys 302 (37). Second, MS/MS fragmentation analysis of the ALDH2 active site peptide identified Cys 302 as the target for 4ONEA modification (see Results section). Although hALDH2 was inhibited by low micromolar 4ONEA, the enzyme did not exhibit affinity for the lipid. At concentrations used (i.e., 1-20 µM), k′ varied linearly with [4ONEA] with no apparent saturation. Such a result demonstrates that hALDH2 inactivation by 4ONEA proceeds as a bimolecular reaction without formation of an enzyme‚inhibitor complex prior to thiol modification via Michael addition (38). This is in agreement with earlier studies that demonstrated that the ALDH enzyme does not have affinity for acid groups and a lack of product inhibition by acids (39). Therefore, inhibition of hALDH2 by the 4ONE-acid product most likely is due to the good nucleophilicity of an active site Cys and notable electrophilicity of 4ONEA, as opposed to product inhibition via affinity for the acid. As shown in Table 1, hALDH2 tryptic peptides other than the one containing the Cys 302 were found to be modified by 4ONE, 4ONEA, and 4HNE (i.e., at 500 µM). The identities of the adducted residues were not determined; however, the majority of peptides modified contained Cys; therefore, it is likely that the target is a thiol given the reactivity toward R,βunsaturated carbonyls. Previous studies have demonstrated reactivity of Cys 162 toward thiol modifiers (40, 41), and this residue could represent a site of adduction for the lipids used in the present study (Table 1). The peptide containing two 4ONE adducts (residues 156-192) has only one free thiol (Cys 162) and a missed trypsin cleavage site (Lys 178); therefore, it is

Aldehyde Dehydrogenase Inhibition by Lipids

likely that the second modification is located on Lys 178, thus preventing tryptic digest at this location. However, His 156 could also represent a viable target. Interestingly, the tryptic peptide containing residues 1-34 was found to contain a 4ONEA adduct (based on mass shift of 170 Da), exclusively, after treatment with 4ONE. This result was also observed in an earlier study utilizing matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (41). The presence of a 4ONEA adduct could be the result of autoxidation of the 4ONE adduct aldehyde to an acid or of modification by 4ONEA produced via hALDH2-catalyzed oxidation of 4ONE as substrate. Perhaps the exiting product, 4ONEA, comes into proximity of a nucleophile on the Nterminal tryptic peptide (e.g., Cys 19). The active site peptide (273-307) was determined to contain either a 4ONE or a 4ONEA adduct, based on mass, after treatment of the hALDH2 protein with 4ONE. Such a finding is likely the result of auto-oxidation of the 4ONE adduct aldehyde to the acid during treatment (30 min) or tryptic digestion (4 h) at 37 °C; however, it is possible that 4ONEA generated in situ could modify the active site thiol. Future experiments to determine whether the thioether linkage between Cys 302 and the 4ONEA adduct is located at the C2 or C3 position could resolve this issue, as 4ONEA is reactive only at C2 while 4ONE can be conjugated at C3 > C2 (15). While modification of Cys residues other than those found in the active site of hALDH2 may affect activity only slightly or not at all, it is conceivable that these adducts could have biological significance, for example, change the rate of protein turnover (33) or alter the ability of hALDH2 to engage in protein-protein interactions. Previous studies have demonstrated the high reactivity of 4ONE toward protein nucleophiles (3), yet resistance of aldehyde-metabolizing enzymes (i.e., alcohol dehydrogenase,3 aldose reductase, and carbonyl reductase) toward inactivation by the lipid aldehyde (16, 17). However, hALDH2 does not fit this trend, but instead, is potently inhibited by both 4ONE and the presumed oxidation product, 4ONEA. Other lipid peroxidation products, such as the endogenously formed 4HNE, malonaldehyde, and acrolein (minor product), have also been found to be good inhibitors of the enzyme (9, 42, 43), thus demonstrating the high sensitivity of hALDH2 to oxidative stress. Such findings are alarming, given the importance of the enzyme in xenobiotic and biogenic amine metabolism (44), and demonstrate the biological necessity of alternative and compensatory pathways for aldehyde biotransformation. In summary, hALDH2 was found to have limited activity toward 4ONE, yielding a reactive oxidation product (i.e., 4ONEA). The GSH conjugate of 4ONE was also found to be a substrate for the enzyme with low turnover but reasonable overall catalytic efficiency. 4ONE and 4ONEA potently and irreversibly inhibited hALDH2, and the mechanism of inactivation was determined to be due to modification of the catalytic nucleophile, Cys 302, via Michael addition by both reactive lipids. These results demonstrate that hALDH2 may not provide an efficient route for 4ONE biotransformation, due to the very limited capacity of the enzyme to catalyze oxidation of the substrate and the sensitivity of hALDH2 toward inhibition by 4ONE and 4ONEA. Instead, the enzyme appears to be a susceptible target for the carbonyl-containing lipids, as is the case for other reactive organic products of oxidative stress. hALDH2 provides an important cellular role in xenobiotic and 3

Unpublished observation.

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biogenic amine metabolism (44); therefore, sustained inhibition of the enzyme by lipid peroxidation products may provide a link between oxidative stress and disease (45). Acknowledgment. This work was supported in part by Grants NIH/NIAAA R01AA09300 and NIH/NIEHS R01ES09410 (D.R.P.), NIH/NIEHS F32ES11937 (J.A.D.), and NIH/NIAAA R01AA11982 (T.D.H.). We thank Z. Kiebler for assistance with managing MS data files and J. Rees for help with NMR analysis.

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