1418
Chem. Res. Toxicol. 2003, 16, 1418-1423
Aldose Reductase Catalyzes Reduction of the Lipid Peroxidation Product 4-Oxonon-2-enal Jonathan A. Doorn,† Satish K. Srivastava,‡ and Dennis R. Petersen*,† Department of Pharmaceutical Sciences, School of Pharmacy, The University of Colorado Health Sciences Center, Denver, Colorado 80262, Department of Human Biological Chemistry and Genetics, The University of Texas Medical Branch, Galveston, Texas 77555 Received August 8, 2003
Recent studies found that 4-oxonon-2-enal (4ONE) is a highly reactive product of lipid peroxidation that can modify peptides and protein sulfhydryls. Because aldose reductase (AR) was shown earlier to catalyze reduction of an R,β-unsaturated lipid aldehyde, 4-hydroxynon2-enal (4HNE), it was systematically investigated if this enzyme could represent a pathway for 4ONE metabolism as well. 4ONE, the glutathione (GSH) conjugate of 4ONE (GS-4ONE), and 1-hydroxynonen-4-one (1HNO), the predicted initial metabolite of 4ONE reduction, were incubated with AR and NADPH, and kinetic constants were measured. The initial product of AR-mediated 4ONE reduction was identified as 1HNO, which could be further reduced to DHN, catalyzed by AR. This result indicates that the order of 4ONE carbonyl reduction is aldehyde and then ketone. 1HNO was found to be an electrophile toward GSH with reactivity ∼55-fold less than 4ONE but ∼2-fold higher than that of 4HNE. The enzyme had activity toward GS4ONE, exhibiting a ∼4-fold higher kcat/KM for GS-4ONE as compared to 4ONE. In the presence of NADPH, 4ONE did not inactivate AR, whereas in the absence of the cofactor, ∼60% of the enzyme activity was lost. The orientation of 4ONE in the AR active site was predicted using molecular modeling to explain the reactivity of 4ONE toward the enzyme. These simulations revealed that concurrent with NADPH binding to AR, Cys 298 is oriented such that the thiol group will not interact with 4ONE. Results of the present study are the first to demonstrate that AR may represent a pathway for metabolism of 4ONE and GS-4ONE.
Introduction AR1 (AKR 1B1; EC 1.1.1.21), a cytosolic enzyme, is a member of the aldo-keto reductase superfamily (1, 2) that is widely expressed in human tissues (3). AR is proposed to participate in the polyol pathway by catalyzing the reduction of glucose to sorbitol in a NADPH-dependent manner (1, 2). However, glucose is a poor substrate for the enzyme, displaying weak affinity for the enzyme (KM ) ∼70 mM), and the catalytic efficiency of the reaction is low (kcat/KM ) 910 M-1 min-1; 4). These investigations indicate that the metabolism of glucose via AR may only occur to a significant degree under conditions of hyperglycemia, and this raises questions as to the physiological role of the enzyme. It was found that AR catalyzes reduction of toxic aldehydes, such as acrolein and 4HNE, generated via cellular oxidative stress and lipid peroxidation, yielding inert alcohols (5-7). Although 4HNE and other lipidderived aldehydes are reactive electrophiles capable of modifying proteins and potently inhibiting enzymes (810), AR is resistant toward inactivation by these chemicals in the presence of NADPH (6). In fact, the lipid* To whom correspondence should be addressed. Tel: (303)315-6159. Fax: (303)315-0274. E-mail:
[email protected]. † The University of Colorado Health Sciences Center. ‡ The University of Texas Medical Branch. 1 Abbreviations: 1HNO, 1-hydroxynon-2-en-4-one; 4HNE, 4-hydroxynon-2-enal; 4ONE, 4-oxonon-2-enal; ADH, alcohol dehydrogenase; AR, aldose reductase; DHN, 1,4-dihydroxynon-2-ene; DMP, Dess-Martin periodinane; DTT, dithiothreitol; GS-4ONE, glutathione-4ONE conjugate; GSH, glutathione.
Figure 1. Lipid peroxidation product 4ONE.
derived aldehydes are the best-known substrates with physiological relevance for the enzyme, implicating a role for AR in detoxifying lipid peroxidation products. 4HNE and other lipid-derived aldehydes that contain an R,β-unsaturated carbonyl undergo spontaneous and enzyme-catalyzed GSH conjugation via Michael addition (11, 12), and the GSH conjugates of these molecules are also substrates for AR (13-15). Interestingly, GSH conjugation of the various lipid aldehydes increases the kcat/KM of AR-catalyzed carbonyl reduction ∼4-1000-fold, as compared to the parent compound. Studies utilizing molecular modeling and site-directed mutagenesis have revealed a GSH-binding site on AR that facilitates efficient accommodation of the GSH-lipid aldehyde conjugate (13-15). Recently, 4ONE (Figure 1) was demonstrated to be a major product of lipid peroxidation (16, 17). This lipid aldehyde is a reactive electrophile capable of modifying DNA and protein side chain nucleophiles (18-21). 4ONE was found to be 10- and 100-fold more reactive toward amino acid/peptide amines and thiols, respectively, as compared to 4HNE (20). Because 4ONE is structurally analogous to 4HNE but more reactive, it may be a suicide
10.1021/tx0300378 CCC: $25.00 © 2003 American Chemical Society Published on Web 09/26/2003
Aldose Reductase Catalyzes Reduction of 4-Oxononenal
substrate for enzymes that metabolize 4HNE, thus increasing the biological half-life of 4HNE and other lipidderived aldehydes. However, the metabolic pathways responsible for detoxification of 4ONE are currently unknown. The present study was undertaken to determine whether reductive catalysis by AR is a potential pathway for 4ONE biotransformation. In the presence of NADPH, recombinant AR efficiently catalyzed the reduction of 4ONE, GS-4ONE, and 1HNO, suggesting an important role of the enzyme in detoxification of this electrophilic biogenic aldehyde. The experiments revealed that substrate reduction was dependent on the binary (ARNADPH) complex because incubation of the enzyme with 4ONE resulted in AR inhibition. Computer-based docking studies performed to determine whether 4ONE is a mechanism-based inhibitor of AR generated models implicating NADPH binding as a critical event associated with reorientation of an active site thiol to a position distant from electrophilic centers of 4ONE.
Materials and Methods Materials. D,L-Glyceraldehyde, DTT, equine liver ADH, GSH, NADH, and NAPDH were purchased from Sigma (St. Louis, MO). DMP and phosphomolybdic acid (20% w/w solution in ethanol) were obtained from Aldrich (Milwaukee, WI). Sephadex G-25 columns (PD-10) were purchased from Amersham Pharmacia Biotech (Uppsala, Sweden). Synthesis of 4ONE and Reduced Derivatives. 4HNE was synthesized as the dimethyl acetal and liberated from the acetal via acid hydrolysis according to procedures described elsewhere (22, 23). 4ONE was prepared by oxidizing 4HNE with DMP (20). 1HNO was synthesized by incubating 4ONE with equine liver ADH and NADH. 1HNO was extracted three times with two volumes of ethyl acetate. The ethyl acetate solution was dried with anhydrous MgSO4 and evaporated to dryness with a gentle stream of nitrogen. Complete conversion of 4ONE to 1HNO was confirmed using TLC (3:1 diethyl ether:n-hexane; 4ONE, Rf ) 0.86; 4HNE, Rf ) 0.61; ADH/4ONE product, Rf ) 0.41) and UV/ vis spectrophotometry (λmax ) 228 and 224 for 4ONE and ADH/ 4ONE product, respectively), and synthesis was confirmed via GC/MS analysis. DHN was synthesized by incubating 4HNE with equine liver ADH and NADH (22, 24). DHN was extracted three times with two volumes of ethyl acetate. The ethyl acetate solution was dried with anhydrous MgSO4 and evaporated to dryness with a gentle stream of nitrogen. Complete conversion of 4HNE to DHN was confirmed using TLC (diethyl ether; 4HNE, Rf ) 0.85; ADH/4HNE product, Rf ) 0.24) and UV/vis spectrophotometry (λmax ) 224 and 202 for 4HNE and ADH/ 4HNE product, respectively), and synthesis was confirmed via GC/MS analysis. Preparation of GSH Conjugates. GS-4ONE was prepared by incubation of 1 mM 4ONE with 1.5 mM GSH in 0.1 M potassium phosphate, pH 7.0, for 30 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 1HNO (GS1HNO) was prepared in a similar manner, except that an incubation time of 60 min was used, and conjugation of 1HNO with GSH was confirmed by an absence of a sharp peak at 224 nm, corresponding to loss of the 1HNO enone system. Enzyme Preparation and Reduction. Recombinant human placental AR (AKR 1B1) was prepared as previously described (25). The enzyme was reduced by incubation with 0.1 M DTT for 1 h at 37 °C in 0.1 M potassium phosphate, pH 7.0, containing 1 mM EDTA. DTT was removed by gel filtration through a PD-10 column assisted by a Bio Rad (Hercules, CA) Econo-Pump using 0.1 M potassium phosphate, pH 7.0, containing 1 mM EDTA. Eluted AR was detected using a Bio Rad
Chem. Res. Toxicol., Vol. 16, No. 11, 2003 1419 Econo-UV Monitor at 280 nm. Fractions containing enzyme were stored on ice under nitrogen and used within 2-3 h. Protein concentration was measured using the BCA assay (Pierce, Rockford, IL; 26). Enzyme Activity. All assays were performed in 0.1 M potassium phosphate, pH 7.0, at 37 °C. Activity was assessed by monitoring the disappearance of NADPH at 340 nm ( ) 6220 M-1 cm-1) using a Molecular Devices SpectraMax 190 plate reader (Sunnyvale, CA). Collected fractions of reduced AR were initially assayed for activity using 2 mM D,L-glyceraldehyde and 0.5 mM NADPH. Lipid aldehyde substrates were used at concentrations varying from 10 to 200 and 10-500 µM for 4ONE and 1HNO, respectively. GSH conjugates of the lipid aldehydes were used at concentrations ranging from 5 to 200 µM for GS4ONE and GS-1HNO. Determination of Enzyme Kinetic Parameters. Enzyme assays were performed in triplicate using six different concentrations of lipid aldehydes and GSH-lipid aldehyde conjugates. Kinetic parameters and statistical analysis were obtained via nonlinear regression analysis of Michael-Menten plots using GraphPad Prism version 3.02 (GraphPad Software, San Diego, CA). Identification of 4ONE-Carbonyl Initially Reduced. Five to eight micrograms of AR was incubated in 0.1 M potassium phosphate, pH 7.0, containing 2 mM 4ONE and 10 mM NADPH for 30 min at 37 °C. Products were extracted three times with two volumes of ethyl acetate. The ethyl acetate solution was dried with MgSO4 and evaporated to dryness with a gentle stream of nitrogen. The reaction product was reconstituted in acetonitrile and stored at -20 °C until analysis. TLC analysis was performed to identify the product using the following conditions and standards: 1:1 n-hexane:ether with standards 4ONE and 4HNE; ether with standards 4ONE, 4HNE, 1HNO, and DHN; and ethyl acetate with standards 1HNO and DHN. To visualize the compounds, the plates were stained with phosphomolybdic acid (20% w/w solution in ethanol) and allowed to dry with gentle heating. Additional confirmation of the reaction product was performed via GC/MS analysis using a HP 5973 GC/MSD system. Determination of Product Reactivity. 1HNO (∼50 µM) was incubated with GSH ranging in concentrations from 0.5 to 5 mM in 50 mM sodium phosphate, pH 7.4, at room temperature (23 ( 1 °C). All experiments were performed using a Molecular Devices SpectraMax 190 plate reader. Change in [1HNO] was determined by monitoring the decrease in absorbance at 224 nm. Because [GSH] was used in g10-fold excess of [1HNO], the bimolecular rate constant (k) could be calculated using pseudofirst-order kinetics (27). k′ was determined from linear regression of ln(% 1HNO) vs time, and k was calculated from the slope of k′ vs [GSH]. Each k′ was calculated from three independent experiments. Linear regression and statistical analysis were performed using Graph Pad Prism version 3.02. Inhibition Experiments of AR with 4ONE. Four treatment groups of AR were prepared in 0.1 M potassium phosphate, pH 7.0: group A, AR alone; group B, AR with 0.5 mM NADPH; group C, AR with 100 µM 4ONE; and group D, AR with 100 µM 4ONE and 0.5 mM NADPH. Incubations were maintained at 37 °C, and activity assays were performed at 30 and 60 min by diluting treatment groups 1:2 with 4 mM D,L-glyceraldehyde and 1 mM NADPH (final concentrations ) 2 and 0.5 mM for D,L-glyceraldehyde and NADPH, respectively) to determine residual activity. Molecular Modeling. An SGI Octane computer (Mountain View, CA) with InsightII 2002 software and Affinity, Biopolymer, Builder, and Discover modules (Accelyris, San Diego, CA) was used for docking experiments. The crystal structure of AR complexed with NADP+ and glucose 6-phosphate (28; 2ACQ) was downloaded from the Brookhaven Protein Data Bank (http://www.rcsb.org/pdb/). Glucose 6-phosphate and crystallographic waters were removed, and the NADP+ was converted to NADPH using the Biopolymer module. Missing residues and hydrogen atoms were added (pH 7.0) with the Biopolymer
1420 Chem. Res. Toxicol., Vol. 16, No. 11, 2003
Doorn et al. Scheme 1
Figure 2. Michaelis-Menten plot for activity of AR/NADPH toward 4ONE. Table 1. Kinetic Constants for AR-Catalyzed Reduction of Substrates
Table 2. Rf Values for TLC Analysis of the Product of AR/NADPH and 4ONE
kinetic parametersa substrateb
Km (µM)
kcat
(min-1)
4ONE 42.0 (( 4.60) 92.2 (( 3.55) 1HNO 17.6 (( 6.71) 8.72 (( 0.747) GS-4ONE 4.62 (( 0.708) 46.1 (( 1.30) GS-1HNO NDc NDc
kcat/Km
Rf valuesa (mM-1
min-1)
2190 (( 120) 495 (( 120) 9950 (( 398) NDc
a Kinetic parameters were determined as described in the Materials and Methods section. All assays were performed in 0.1 M potassium phosphate buffer, pH 7.0, at 37 °C. b Substrates were synthesized as described in the Materials and Methods section. c ND, not determined. No activity was detected with GS-1HNO.
module. Using the Builder module, 4ONE was generated and manually docked in the active site of AR. The aldehyde oxygen of 4ONE was tethered to the hydroxyl of Tyr 48, which serves as the proton donor in the reaction (14). 4ONE and a subset of AR atoms located within a 10 Å radius were designated as flexible, while the rest of the enzyme was held rigid. 4ONE was confined to a 2 Å radius to prevent it from drifting out of the active site. Docking was accomplished via a two phase energy minimization process followed by a molecular dynamics simulation and a final round of energy minimization. With the exception of the initial phase of energy minimization, a distancedependent dielectric was used ( ) 5.0) to simulate solvation by water.
Results Kinetic Characterization of AR with 4ONE and 1HNO. AR was incubated with NADPH and substrates, and the initial velocity was assessed by monitoring a linear decrease in absorbance at 340 nm, corresponding to enzyme-catalyzed oxidation of the cofactor to NADP+. Kinetic data show that AR efficiently catalyzes reduction of 4ONE, with values for kcat and KM of 92.2 min-1 and 42.0 µM, respectively (Figure 2 and Table 1). The predicted metabolite, 1HNO, was also determined to be a substrate for the enzyme (Table 1). As compared to 4ONE, the KM for 1HNO was significantly lower (i.e., ∼60%); however, the kcat and catalytic efficiency were also significantly lower (i.e., ∼90 and 80%, respectively). Collectively, these results indicate that AR efficiently catalyzes the reduction of both carbonyl groups in 4ONE. Identification of 4ONE-Carbonyl Initially Reduced. 4ONE was added to AR and NADPH at pH 7.4, 37 °C, with [4ONE]final ) 2.0 mM, and allowed to react for 30 min. The initial product was isolated by solvent extraction using ethyl acetate and identified via TLC and GC/MS analysis (see Scheme 1). The Rf of the reaction product corresponded to synthesized 1HNO (Table 2). No band corresponding to 4HNE was observed in the solvent extract. The DHN produced was below the detection limit.
compoundb
solvent Ac
4ONE 4HNE 1HNO DHN reaction product
0.68 0.31 0.16
solvent Bd
solvent Ce
0.79 0.46 0.79
0.91 0.67 0.44 0.13 0.44
a
TLC analysis performed and Rf values determined as described in the Materials and Methods section. b Standards synthesized and initial reaction product obtained as described in the Materials and Methods section. c Solvent A ) 1:1 hexane:ether. d Solvent B ) ethyl acetate. e Solvent C ) ether. Table 3. Rate Constants for GSH Conjugation 4ONEb 1HNO 4HNEb
k (M-1 s-1)a
k4ONE/kaldehyde
145 (( 10.1) 2.62 (( 0.324) 1.33 (( 0.0500)
1.0 55 110
a Rate constant for conjugation of GSH with 1HNO was determined as described in the Materials and Methods section. The reaction was carried out in 50 mM sodium phosphate buffer, pH 7.4, at 23 ((1) °C. b Ref 19. Reactions performed in 50 mM sodium phosphate buffer, pH 7.4, at 23 ((1) °C.
GC/MS analysis confirmed the identity of the initial reaction product as 1HNO (see Scheme 1). Fragmentation of the reaction product yielded the following major ions at m/z: 57, 72, 85, 100, and 125. Comparison of these values with m/z for major fragments of 4ONE, 4HNE, and a synthesized standard of 1HNO confirmed that the initial reaction product is 1HNO, the result of 4ONEaldehyde reduction. Initial Reaction Product 1HNO Is an Electrophile. The electrophilic properties of 4ONE suggest that enzymatic reduction by AR may in fact produce products that retain significant reactivity toward nucleophiles. This notion was confirmed by experiments that demonstrated 1HNO to be reactive toward GSH, as indicated by loss of absorbance at 224 nm when the two chemicals were coincubated. Pseudo-first-order kinetics were observed under the conditions used (i.e., [GSH] in g10-fold excess over [1HNO]), indicating that the conjugation reaction is second-order as expected. The bimolecular rate constant (k) for modification of GSH by 1HNO was determined to be 2.62 M-1 s-1 (Table 3). The comparison of this k with that previously determined for 4ONE and 4HNE as shown in Table 3 clearly reveals the striking reactivity of 4ONE with GSH as compared to the rate of GSH conjugation by 4HNE and 1HNO. AR Catalyzes Reduction of GS-4ONE. To evaluate the possibility that GS-4ONE is also a substrate for AR,
Aldose Reductase Catalyzes Reduction of 4-Oxononenal
the GSH conjugate of 4ONE was prepared and incubated with AR and NADPH. GS-4ONE was found to be an excellent substrate for AR, as evident by a linear decrease in absorbance at 340 nm corresponding to enzymecatalyzed oxidation of cofactor to NADP+. kcat and KM were measured to be 46.1 min-1 and 4.62 µM, respectively, yielding a kcat/KM of 9950 mM-1 min-1 (Table 1). The catalytic efficiency of GS-4ONE reduction by AR is ∼4-fold higher than that of 4ONE, indicating that conjugation of 4ONE with GSH results in a product (GS4ONE) that is a better substrate for AR than the parent compound. AR Is Resistant toward Inactivation by 4ONE. Because of the electrophilic nature of 4ONE, it was of interest to determine if 4ONE interacted with any susceptible nucleophilc residues in or adjacent to the active site of AR. Although the AR active site contains a free thiol (i.e., Cys 298) that could be modified by 4ONE resulting in loss of activity (6), the enzyme was found to be resistant toward 4ONE in the presence of NADPH. AR incubated with 0.5 mM NADPH and 100 µM 4ONE for 30 or 60 min at 37 °C resulted in no inhibition of activity toward D,L-glyceraldehyde. At 30 and 60 min, activity was 97.4 (( 11.4) and 104 (8.09)%, respectively, as compared to a control. Treatment of the enzyme with 100 µM 4ONE in the absence of cofactor resulted in loss of 37.1 (( 1.97) and 64.5 (( 8.91)% activity at 30 and 60 min, respectively, toward the substrate D,L-glyceraldehyde. Active Site Cys 298 in the Binary Complex of AR with NADPH Is Not Reactive toward 4ONE. Molecular modeling of 4ONE in the active site of AR complexed with NAPDH was performed to determine why the enzyme is resistant toward inhibition by the reactive electrophile 4ONE. It was found that 4ONE oriented itself in the AR active site such that the thiol moiety of Cys 298 was not in proximity to react with the R,βunsaturated carbonyls of 4ONE, specifically C2 and C3 of the lipid aldehyde (Figure 3). For all three of the structures shown, the Cys 298 thiol was measured to be >9 Å from C2 or C3 of the lipid aldehyde. Furthermore, Trp 20 and Trp 219 may sterically hinder the reaction of Cys 298 with 4ONE. The results from these molecular modeling studies provide rational explanations for the resistance of the binary complex of AR (AR-NADPH) to inactivation by electrophilic interactions with 4ONE.
Discussion Previously, studies reported a KM value of AR for 4HNE ranging from ∼10 to 30 µM (5, 6, 13, 29). In the present study, a similar result was found for the structurally analogous 4ONE, with KM ) 42.0 µM. Oxidation of the hydroxyl at C4 to a ketone does not change the KM of AR for the substrate, indicating that the enzyme does not make specific contact (e.g., hydrogen bonding) with the functional moiety at C4 (i.e., hydroxyl for 4HNE and ketone for 4ONE). According to the solved crystal structure, the AR active site lacks polar residues that could form hydrogen bonds with substrates (30), and this may explain why the KM of the enzyme for 4HNE and 4ONE is comparable. The kcat measured for 4ONE (i.e., 92 min-1) is within the general range of values reported for other aldehyde substrates (6, 13, 29) and is consistent with the proposal that E‚NADP isomerization is the rate-limiting step for
Chem. Res. Toxicol., Vol. 16, No. 11, 2003 1421
Figure 3. Models of 4ONE in the active site of AR complexed with NADPH generated via computer-assisted docking experiments. Shown are the three lowest energy structures generated. For the sake of clarity, only a select number of residues in the active site of AR are shown, and protons were omitted. The aldehyde carbonyl of 4ONE is displayed as a ball-and-stick structure. Atoms were given the following color assignments: carbon, gray; nitrogen, green; oxygen, red; and sulfur, yellow. The nicotinamide ring of NADPH is located directly below the 4ONE aldehyde. For both structures shown, Cys 298 is predicted to be distant from C2 and C3 of 4ONE (i.e., >9 Å) and sterically hindered by Trp 20 and Trp 219, thus explaining the lack of reactivity of this thiol toward the lipid aldehyde.
the overall reaction (31, 32). However, 4ONE turnover is ∼3-5-fold higher than that for 4HNE reported in some previous studies (5, 6, 13). The observed disparity could be due to differences in reaction temperatures of experiments (i.e., 25 vs 37 °C used in the present study). The results presented here demonstrate that the initial product of AR-catalyzed hydride transfer to 4ONE is 1HNO (Scheme 1). Because 1HNO contains an R,βunsaturated ketone, it was predicted to be a reactive electrophile capable of modifying protein thiols and amines and requiring additional metabolism (e.g., enzymecatalyzed reduction, GSH conjugation). The novel results described here show that 1HNO is also a substrate for AR, with a KM and kcat of 17.6 µM and 8.72 min-1, respectively. This finding demonstrates that AR is capable of catalyzing the reduction of both carbonyls of 4ONE to yield the inert DHN (Scheme 1). Such a result is analogous to methyl glyoxal metabolism, in which both methyl glyoxal and the major reduced product acetol (1hydroxy acetone) are substrates for AR (33). However, the turnover and catalytic efficiency for 1HNO reduction are ∼10- and 5-fold less, respectively, than that for 4ONE. Therefore, 4ONE is the preferred substrate for AR as compared to 1HNO and may competitively inhibit enzyme-catalyzed reduction of 1HNO. Although the enzyme can catalyze reduction of a ketone as evident by activity toward 1HNO (Table 1), the fact that 1HNO but not 4HNE was observed as the initial product demonstrates that AR mediates hydride transfer specifically to the aldehyde of 4ONE. Such a result has been previously observed for glyoxals, where AR preferentially catalyzes reduction of the aldehyde (33) unless GSH is present to form a thiohemiacetal with the aldehyde, resulting in the ketone being the hydride acceptor (34). DHN generated was below the detection
1422 Chem. Res. Toxicol., Vol. 16, No. 11, 2003
limit after the specified time period for the reaction. The absence of detectable DHN might be the result of competitive inhibition of AR-catalyzed 1HNO reduction by residual 4ONE, calculated to be at a concentration of 1.4-1.6 mM using the Vmax parameter shown in Table 1. Furthermore, the turnover for 1HNO is >10-fold lower than that for 4ONE (Table 1). The initial reaction product 1HNO was biosynthesized using ADH, NADH, and 4ONE and then incubated with GSH to determine its reactivity toward nucleophiles. 1HNO was found to be an electrophile that modified GSH with a bimolecular rate constant (k) of 2.61 M-1 s-1. This value is ∼55-fold less than that previously determined for GSH with 4ONE, demonstrating that AR-catalyzed carbonyl reduction of 4ONE results in a significantly less reactive product (20). However, on the basis of rate constants, 1HNO is twice as reactive toward GSH as 4HNE, which is considered to be a potent electrophile capable of modifying cellular nucleophiles (11, 20). The reduction of 4ONE by AR/NADPH, therefore, does not initially yield an inert compound but, instead, results in a product that can modify proteins and may require additional metabolism before being inactivated. 4ONE metabolism may represent a complex problem because both the parent compound and the metabolites are able to react with cellular nucleophiles, yielding covalent adducts. Previous studies have reported that conjugation of GSH with a lipid aldehyde (e.g., acrolein) increases the catalytic efficiency of AR-catalyzed reduction of the aldehydic substrate by a factor of ∼4-1000-fold, and this effect is the result of a specific GSH-binding region in the active site of AR (13-15). A similar result was observed in the present study (Table 1 and Scheme 1). The GS-4ONE conjugate was found to be a substrate for AR, with kcat and KM of 46.1 min-1 and 4.62 µM. As compared to 4ONE, GS-4ONE has a ∼4-fold higher kcat/ KM, indicating that it is a superior substrate for the enzyme. However, it should be noted that GSH can theoretically react via Michael addition with either C2 or C3 of 4ONE, yielding two structural isomers. Because the Michael reaction can yield two chiral products, each structural isomer will have two stereoisomers. Therefore, the measured kinetic parameters may be a composite function of the kinetic constants for each structural isomer and stereoisomer of GS-4ONE. Yet, these results suggest that the GSH-binding motif of AR accommodates interactions of the enzyme with GSH conjugates of small hydrophilic R,β-unsaturated aldehydes (i.e., acrolein) as well as larger, more hydrophobic aldehydes such as 4ONE. 4ONE is a reactive electrophile that can modify protein residues such as Arg, Cys, His, and Lys (20, 21). However, AR was found to be resistant toward 4ONEmediated enzyme inactivation in the presence of NADPH. AR treated with 100 µM 4ONE at 37 °C for 60 min retained 35% D,L-glyceraldehyde activity relative to a control. When 0.5 mM NADPH was included in the reaction mixture, no enzyme activity was lost over the course of 60 min. The reaction of AR and NADPH with 4ONE will yield the product 1HNO, which was shown in the present study to be a reactive electrophile (Table 3). The fact that no inhibition of activity was observed for enzyme incubated with 4ONE and NADPH indicates that AR is also resistant toward the product 1HNO. These results are consistent with a previous study that reported AR to be completely resistant toward lipid
Doorn et al.
aldehydes (e.g., 4HNE) in the presence of NADPH and to lose partial activity in the absence of cofactor (6). The active site of AR contains three Cys residues (i.e., Cys 80, 298, and 303). However, only Cys 298 modification affects the enzyme activity. Adduction of this residue by small molecular weight compounds such as nitric oxide activates the enzyme whereas modification by larger molecules such as GSSG and HNE significantly inactivate the enzyme. Although 4ONE is a more potent (>100fold) modifier of Cys than 4HNE (20), it is not clear why AR is not completely inhibited by a high concentration of 4ONE (i.e., 100 µM). It is possible that Cys 298 is not a reactive nucleophile for the following reasons. First, the solved AR crystal structure revealed that the enzyme active site is mainly comprised of hydrophobic residues (30). A lack of polar residues to act as acceptors for the thiol proton as well as charged residues that can polarize the sulfhydryl group may severely diminish reactivity of the Cys 298 (20, 34-36). Furthermore, Michael addition is acid catalyzed (37); therefore, the lack of proton donors in the active site of AR may inhibit reaction of the Cys 298 thiol with an R,β-unsaturated carbonyl. Second, computer-assisted docking experiments, involving molecular mechanics and dynamics, performed in the present study revealed that 4ONE may be oriented in the AR active site such that it is not in proximity to react with Cys 298. The thiol of Cys 298 is predicted to be >9 Å in distance from C2 or C3 of 4ONE (Figure 3). Furthermore, Cys 298 was calculated to be “buried” behind several bulky hydrophobic residues (e.g., Trp 20 and Trp 219), thus implicating steric hindrance as a factor in the lack of reactivity for this residue. In summary, the novel results presented here demonstrate that AR efficiently catalyzes reduction of the lipid peroxidation product 4ONE as well as derivatives of the lipid aldehyde as depicted in Scheme 1. The initial reaction of enzyme with NADPH and 4ONE resulted specifically in aldehyde reduction, yielding 1HNO. It was also demonstrated that AR exhibited activity toward the initial product, 1HNO, indicating that the enzyme is capable of fully metabolizing 4ONE to the inert DHN. Consistent with previous papers (13-15), GS-4ONE was found to be a superior substrate as compared to the parent compound 4ONE. Even though 4ONE is a reactive electrophile capable of modifying protein residues, AR in the presence of NADPH is resistant toward inactivation by the lipid aldehyde because cofactor confers complete protection of the enzyme. Taken together, these results indicate that AR is potentially an integral enzyme involved in the biotransformation of the potentially cytotoxic lipid peroxidation product 4ONE and its chemically reactive derivatives.
Acknowledgment. This work was supported in part by Grants NIH/NIAAA R01AA09300 and NIH/NIEHS R01ES09410 (D.R.P), NIH/NIEHS F32 ES11937 (J.A.D), and DK 36118 (S.K.S.). We thank Dr. D. Claffey, Department of Pharmaceutical Sciences, University of Colorado Health Sciences Center, for performing the GC/MS analysis. Mass spectral analysis was performed at the Biochemical Mass Spectrometry Core Facility, the University of Colorado Health Sciences Center.
References (1) Bhatnagar, A., and Srivastava, S. K. (1992) Aldose reductase: congenial and injurious profiles of an enigmatic enzyme. Biochem. Med. Metab. Biol. 48, 91-121.
Aldose Reductase Catalyzes Reduction of 4-Oxononenal (2) Yabe-Nishimura, C. (1998) Aldose reductase in glucose toxicity: a potential target for the prevention of diabetic complications. Pharmacol. Rev. 31, 21-33. (3) O’Connor, T., Ireland, L. S., Harrison, D. J., and Hayes, J. D. (1999) Major differences exist in the function and tissue-specific expression of human B1 aldehyde reductase and the principal human aldo-keto reductase AKR1 family members. Biochem. J. 343, 487-504. (4) Vander Jagt, D. L., Hunsaker, L. A., Robinson, B., Stangebye, L. A., and Deck, L. M. (1990) Aldose reductase from human skeletal and heart muscle. Interconvertible forms related by thiol-disulfide exchange. J. Biol. Chem. 19, 10912-10918. (5) Srivastava, S., Chandra, A., Bhatnagar, A., Srivastava, S. K., and Ansari, N. (1995) Lipid peroxidation product, 4-hydroxynonenal and its conjugate with GSH are excellent substrates of bovine lens aldose reductase. Biochem. Biophys. Res. Commun. 217, 741-746. (6) Srivastava, S., Watowich, S. J., Petrash, J. M., Srivastava, S. K., and Bhatnagar, A. (1999) Structural and kinetic determinants of aldehyde reduction by aldose reductase. Biochemistry 38, 4254. (7) Srivastava, S., Chandra, A., Wang, L. F., Seifert, W. E., Jr., DaGue, B. B., Ansari, N. H., Srivastava, S. K., and Bhatnagar, A. (1998) Metabolism of the lipid peroxidation product, 4-hydroxytrans-2-nonenal, in isolated perfused rat heart. J. Biol. Chem. 273, 10893-10900. (8) Ji, C., Kozak, K. R., and Marnett, L. J. (2001) IκB kinase, a molecular target for inhibition by 4-hydroxy-2-nonenal. J. Biol. Chem. 276, 18223-18228. (9) Jurgens, G., Lang, J., and Esterbauer, H. (1986) Modification of human low-density lipoprotein by the lipid peroxidation product 4-hydroxynonenal. Biochim. Biophys. Acta 875, 103-114. (10) Nguyen, E., and Picklo, M. J., Sr. (2003) Inhibition of succinic semialdehyde dehydrogenase activity by alkenal products of lipid peroxidation. Biochim. Biophys. Acta 1637, 107-112. (11) Esterbauer, H., Schaur, R. J., and Zollner, H. (1991) Chemistry and biochemistry of 4-hydroxynonenal, malonaldehyde and related aldehydes. Free Radical Biol. Med. 11, 81-128. (12) Alin, P., Danielson, U. H., and Mannervik, B. (1985) 4-Hydroxyalk-2-enals are substrates for glutathione transferase. FEBS Lett. 179, 267-270. (13) Ramana, K. V., Dixit, B. L., Srivastava, S., Balendiran, G. K., Srivastava, S. K., and Bhatnagar, A. (2000) Selective recognition of glutathiolated aldehydes by aldose reductase. Biochemistry 39, 12172-12180. (14) Dixit, B. L., Balendiran, G. K., Watowich, S. J., Srivastava, S., Ramana, K. V., Petrash, J. M., Bhatnagar, A., and Srivastava, S. K. (2000) Kinetic and structural characterization of the glutathione-binding site of aldose reductase. J. Biol. Chem. 275, 21587-21595. (15) Ramana, K. V., Dixit, B. L., Srivastava, S., Bhatnagar, A., Balendiran, G. K., Watowich, S. J., Petrash, J. M., and Srivastava, S. K. (2001) Characterization of the glutathione binding site of aldose reductase. Chem.-Biol. Interact. 130-132, 537-548. (16) Lee, S. H., and Blair, I. A. (2000) Characterization of 4-oxo-2nonenal as a novel product of lipid peroxidation. Chem. Res. Toxicol. 13, 698-702. (17) Lee, S. H., Oe, T., and Blair, I. A. (2001) Vitamin C-induced decomposition of lipid hydroperoxides to endogenous genotoxins. Science 292, 2083-2086. (18) Lee, S. H., Rindgen, D., Bible, R. H., Jr., Hajdu, E., and Blair, I. A. (2000) Characterization of 2′-deoxyadenosine adducts derived from 4-oxo-2-nonenal, a novel product of lipid peroxidation. Chem. Res. Toxicol. 13, 565-574. (19) Rindgen, D., Lee, S. H., Nakajima, M., and Blair, I. A. (2000) Formation of a substituted 1,N(6)-etheno-2′-deoxyadenosine adduct by lipid hydroperoxide-mediated generation of 4-oxo-2nonenal. Chem. Res. Toxicol. 13, 846-852. (20) Doorn, J. A., and Petersen, D. R. (2002) Covalent modification of amino acid nucleophiles by the lipid peroxidation products
Chem. Res. Toxicol., Vol. 16, No. 11, 2003 1423
(21)
(22)
(23)
(24)
(25)
(26)
(27) (28)
(29)
(30)
(31)
(32)
(33)
(34)
(35)
(36)
(37)
4-hydroxy-2-nonenal and 4-oxo-2-nonenal. Chem. Res. Toxicol. 15, 1445-1450. Zhang, W.-H., Liu, J., Xu, G., Yuan, G., and Sayre, L. M. (2003) Model studies on protein side chain modification by 4-oxo-2nonenal. Chem. Res. Toxicol. 16, 512-523. Esterbauer, H., Zollner, H., and Lang, J. (1985) Metabolism of the lipid peroxidation product 4-hydroxynonenal by isolated hepatocytes and by liver cytosolic fractions. Biochem. J. 228, 363373. Esterbauer, H., and Weger, W. (1967) U ¨ ber die wirkungen von aldehyden auf gesunde und maligne zellen, 3. mitt: Synthese von homologen 4-hydroxy-2-alkenalen, II. Monatsh. Chem. 98, 19942000. Hartley, D. P., Ruth, J. A., and Petersen, D. R. (1995) The hepatocellular metabolism of 4-hydroxynonenal by alcohol dehydrogenase, aldehyde dehydrogenase, and glutathione S-transferase. Arch. Biochem. Biophys. 316, 197-205. Petrash, J. M., Harter, T. M., Devine, C. S., Olins, P. O., Bhatnagar, A., Liu, S., and Srivastava, S. K. (1992) Involvement of cysteine residues in catalysis and inhibition of human aldose reductase. Site-directed mutagenesis of Cys-80, -298, and -303. J. Biol. Chem. 267, 24833-24840. Smith, P. K., Krohn, R. I., Hermanson, G. T., Mallia, A. K., Gartner, F. H., Provenzano, M. D., Fujimoto, E. K., Goeke, N. M., Olson, B. J., and Klenk, D. C. (1985) Measurement of protein using bicinchoninic acid. Anal. Biochem. 150, 76-85. Atkins, P. W. (1995) Physical Chemistry, 5th ed., W. H. Freeman and Co., New York. Harrison, D. H., Bohren, K. M., Ringe, D., Petsko, G. A., and Gabbay, K. H. (1994) An anion binding site in human aldose reductase: mechanistic implications for the binding of citrate, cacodylate, and glucose 6-phosphate. Biochemistry 33, 2011-2020. Vander Jagt, D. L., Kolb, N. S., Vander Jagt, T. J., Chino, J., Martinez, F. J., Hunsaker, L. A., and Royer, R. E. (1995) Substrate specificity of human aldose reductase: identification of 4-hydroxynonenal as an endogenous substrate. Biochim. Biophys. Acta 1249, 117-126. Wilson, D. K., Bohren, K. M., Gabbay, K. H., and Quiocho, F. A. (1992) An unlikely sugar substrate site in the 1.65 A structure of the human aldose reductase holoenzyme implicated in diabetic complications. Science 257, 81-84. Grimshaw, C. E. (1992) Aldose reductase: model for a new paradigm of enzymic perfection in detoxification catalysts. Biochemistry 31, 10139-10145. Kubiseski, T. J., Hyndman, D. J., Morjana, N. A., and Flynn, T. G. (1992) Studies on pig muscle aldose reductase. Kinetic mechanism and evidence for a slow conformational change upon coenzyme binding. J. Biol. Chem. 267, 6510-6517. Vander Jagt, D. L., Hassebrook, R. K., Hunsaker, L. A., Brown, W. M., and Royer, R. E. (2001) Metabolism of the 2-oxoaldehyde methylglyoxal by aldose reductase and by glyoxalase-I: roles for glutathione in both enzymes and implications for diabetic complications. Chem.-Biol. Interact. 130-132, 549-562. Snyder, G. H., Cennerazzo, M. J., Karalis, A. J., and Field, D. (1981) Electrostatic influence of local cysteine environments on disulfide exchange kinetics. Biochemistry 20, 6509-6519. Dinkova-Kostova, A. T., Massiah, M. A., Bozak, R. E., Hicks, R. J., and Talalay, P. (2001) Direct evidence that sulfhydryl groups of Keap1 are the sensors regulating induction of phase 2 enzymes that protect against carcinogens and oxidants. Proc. Natl. Acad. Sci. U.S.A. 98, 3404-3409. Lutolf, M. P., Tirelli, N., Cerritelli, S., Cavalli, L., and Hubbell, J. A. (2001) Systematic modulation of Michael-type reactivity of thiols through the use of charged amino acids. Bioconjugate Chem. 12, 1051-1056. Wade, L. G., Jr. (2003) Organic Chemistry, 5th ed., Prentice Hall, Upper Saddle River, NJ.
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