Partially irreversible inactivation of mitochondrial aldehyde dehydrogenase by nitroglycerin

Abstract

Mitochondrial aldehyde dehydrogenase (ALDH2) may be involved in the biotransformation of glyceryl trinitrate (GTN), and the inactivation of ALDH2 by GTN may contribute to the phenomenon of nitrate tolerance. We studied the GTN-induced inactivation of ALDH2 by UV/visible absorption spectroscopy. Dehydrogenation of acetaldehyde and hydrolysis of p-nitrophenylacetate (p-NPA) were both inhibited by GTN. The rate of inhibition increased with the GTN concentration and decreased with the substrate concentration, indicative of competition between GTN and the substrates. Inactivation of p-NPA hydrolysis was greatly enhanced in the presence of NAD(+), and, to a lesser extent, in the presence of NADH. In the presence of dithiothreitol (DTT) inactivation of ALDH2 was much slower. Dihydrolipoic acid (LPA-H(2)) was less effective than DTT, whereas glutathione, cysteine, and ascorbate did not protect against inactivation. When DTT was added after complete inactivation, dehydrogenase reactivation was quite modest (< or =16%). The restored dehydrogenase activity correlated inversely with the GTN concentration but was hardly affected by the concentrations of acetaldehyde or DTT. Partial reactivation of dehydrogenation was also accomplished by LPA-H(2) but not by GSH. We conclude that, in addition to the previously documented reversible inhibition by GTN that can be ascribed to the oxidation of the active site thiol, there is an irreversible component to ALDH inactivation. Importantly, ALDH2-catalyzed GTN reduction was partly inactivated by preincubation with GTN, suggesting that the inactivation of GTN reduction is also partly irreversible. These observations are consistent with a significant role for irreversible inactivation of ALDH2 in the development of nitrate tolerance.

FIGURE 1.

Inhibition by GTN of ALDH2-catalyzed dehydrogenation of acetaldehyde. Panel A shows a time trace for the formation of NADH from NAD⁺, monitored at 340 nm. At t = 0 the cuvette contained 0.2 mm acetaldehyde and 0.2 mm NAD⁺ in 50 mm potassium P_i (pH 7.4). At t = 80 s, catalysis was initiated by the addition of 33 μg/ml ALDH2. Inactivation started at t = 480 s by the addition of 0.05 mm GTN. After inactivation of the enzyme, at t = 1160 s, an attempt was made to restore activity by the addition of 1 mm DTT. The dots represent the data points, whereas the continuous lines are best fits to the data. Linear fits were applied to the phases before (no catalysis (-0.10 ± 0.06) × 10^-4 absorbance units (a.u.)/s), and after ALDH2 addition (initial activity, (8.03 ± 0.05) × 10^-4 a.u./s), and to the activity after DTT admission (restored activity (1.378 ± 0.006) × 10^-4 a.u./s); for the inactivation after GTN addition a combination of a single exponential and a linear fit was applied (apparent inactivation rate constant (7.77 ± 0.06) × 10^-3 s^-1; residual activity (0.133 ± 0.007) × 10^-4 a.u./s). Panel B compares the residual and restored rates of acetaldehyde dehydrogenation after addition of GTN and DTT, respectively. Experimental conditions: 33 μg/ml ALDH2, 0.43 mm acetaldehyde, 0.4 mm NAD⁺, 0.4 mm DTT, and concentrations of GTN as indicated in 50 mm potassium P_i (pH 7.4). Initial dehydrogenase activities under the conditions applied here amounted to 289 ± 13 nmol min^-1 mg^-1, which corresponds to a turnover number of 69 ± 3 min^-1.

FIGURE 2.

Effects of thiols on the inactivation of dehydrogenation by GTN. Panel A, restoration by different thiols of ALDH2-catalyzed dehydrogenation after inactivation by GTN. Plotted are the relative residual activities (v_residual/v_initial) after inactivation by 0.1 mm GTN and the relative restored activities (v_restored/v_initial) after addition of 1 mm DTT, LPA-H₂, or GSH. Initial conditions: 25 μg/ml ALDH2, 0.45 mm acetaldehyde, and 0.4 mm NAD⁺ in 0.2 m potassium P_i (pH 7.4). Panel B, protection by DTT of dehydrogenase activity against GTN-mediated inactivation. The figure shows representative time traces of the ALDH2-catalyzed formation of NADH, as monitored at 340 nm before and after addition of GTN in the presence or absence of DTT. Experimental conditions: 33 μg/ml ALDH2, 0.45 mm acetaldehyde, 0.4 mm NAD⁺, and 0.1 mm GTN with or without 1 mm DTT in 50 mm potassium P_i (pH 7.4).

FIGURE 3.

Inhibition by GTN of ALDH2-catalyzed hydrolysis of p-nitrophenylacetate. The figure shows the formation of p-nitrophenol from p-NPA, as monitored at 400 nm. Panel A shows that, in the absence of NAD⁺, GTN did not significantly affect esterase activity. However, inactivation set in immediately after the addition of NAD⁺. Experimental conditions were: p-NPA (0.2 mm) in 50 mm potassium P_i (pH 7.4) was present at the start of the reaction; ALDH2 (33 μg/ml), GTN (0.1 mm), DTT (0.2 mm), and NAD⁺ (0.2 mm) were added as indicated. Curve fitting as described in the legend to Fig. 1 yielded (1.0 ± 0.5) × 10^-5 absorbance units (a.u.)/s for the uncatalyzed reaction, (1.07 ± 0.02) × 10^-3 a.u./s for the initial rate, (1.083 ± 0.008) × 10^-3 a.u./s and (1.15 ± 0.01) × 10^-3 a.u./s after addition of GTN and DDT, respectively, (1.051 ± 0.008) × 10^-2 s^-1 for the apparent inactivation rate constant after addition of NAD⁺, and (7.2 ± 0.1) × 10^-5 a.u./s for the residual activity. Panel B demonstrates that GTN inactivates the enzyme in the presence of NAD⁺. Experimental conditions: p-NPA (0.2 mm) and NAD⁺ (0.2 mm) in 50 mm potassium P_i (pH 7.4) were present at the start of the reaction; ALDH2 (33 μg/ml) and GTN (0.1 mm) were added as indicated. Curve fitting yielded the following results: (0.048 ± 0.009) × 10^-3 a.u./s for the uncatalyzed reaction, (3.65 ± 0.08) × 10^-3 a.u./s for the initial activity, (2.6 ± 0.1) × 10^-2 s^-1 for the apparent inactivation rate constant, and (0.0567 ± 0.0008) × 10^-3 a.u./s for the residual activity.

FIGURE 4.

Effect of NAD⁺ and NADH on the rate of inactivation by GTN of ester hydrolysis. The figure shows the effects of NAD⁺ and NADH (0.2 mm) on the apparent inactivation rate constant in the absence and presence of 0.4 mm DTT. Results are presented as averages ± S.E. (n = 3). Further conditions: 33 μg/ml ALDH2, 0.2 mm p-NPA, and 0.1 mm GTN in 50 mm potassium P_i (pH 7.4).

FIGURE 5.

Effect of preincubation of ALDH2 with GTN on acetaldehyde dehydrogenation. The figure shows the effects of preincubation of ALDH2 at pH 7.5 with 1 μm GTN on the subsequent determination of dehydrogenase activity in the presence of NAD⁺ at pH 7.4 in the absence and presence of 2 mm DTT and LPA-H₂. Experimental conditions for preincubation were: 0.27 mg/ml ALDH2, 1 μm GTN, 0.2 mm MgCl₂, 2 mm NAD⁺, 50 mm NaP_i (pH 7.5) for 10 min at 37 °C. Assay conditions were: 1.5 μg/ml ALDH2, 2 mm NAD⁺, 10 mm MgCl₂, and 0.2 mm acetaldehyde in 50 mm NaP_i (pH 7.5) at 25 °C. See “Experimental Procedures” for further details.

FIGURE 6.

Effect of preincubation time on the dehydrogenase activity of ALDH2. The enzyme was preincubated for various times, after which dehydrogenase activities were determined by monitoring the linear absorbance increase at 340 nm over a period of 5 min. Preincubation was performed in the absence (open symbols) and presence (filled symbols) of DTT, and in the absence (circles) or presence (squares) of GTN. Preincubation conditions were: 0.48 mg/ml ALDH2, 1 mm NAD⁺, and 0.18 mm GTN and 0.2 mm DTT as indicated in 300 mm potassium P_i (pH 7.4). Assay conditions were: 19 μg/ml ALDH2, 1 mm acetaldehyde, 1 mm NAD⁺, and 2 mm DTT in 50 mm potassium P_i (pH 7.4). Both incubation and preincubation were performed at 20 °C.

FIGURE 7.

Effect of preincubation of ALDH2 with GTN on the ALDH2-catalyzed reduction of GTN. GTN reduction was determined in the absence or presence of DTT (pre-column activity). Subsequently, the enzyme was rapidly recovered from the assay mixture by YM-10 chromatography, and the GTN reductase activity was measured again in the absence or presence of thiols (post-column activity). Experimental conditions were: 4 μg of ALDH2, 2 μm [¹⁴C]GTN (50 000 dpm), 3 mm MgCl₂, 1 mm NAD⁺, 1 mm EDTA, 1 mm EGTA, and 2 mm GSH with 2 mm DTT as indicated in 50 mm potassium P_i (pH 7.4) for 10 min at 37 °C. See “Experimental Procedures” for further details.

FIGURE 8.

Inhibition by GTN and reactivation by thiols of ALDH in intact mitochondria. The figure depicts the effect of preincubation with GTN (0.1 mm, 10 min, 37 °C) on the dehydrogenation of Monal 62 in the absence and presence of various thiols. Experimental conditions were: 8.1 μg/ml rat liver mitochondria, 3 μm Monal 62, 0.1 mm NAD⁺, and DTT (2 mm), β-ME (50 mm), or LPA-H₂ (0.1 mm) as indicated, in 10 mm Tris (pH 7.4), 250 mm sucrose, 5 mm EGTA, and 2 mm MgCl₂.

SCHEME 1.

Irreversible inactivation of ALDH-catalyzed GTN reduction. Under low turnover conditions ([GTN], [DTT] << Km^GTN, Km^DTT), the enzyme cycles between the reduced thiol/thiolate, oxidized thiyl, and oxidized disulfide states. At higher concentrations, GTN and the thiol reductant (here represented by DTT) compete for the oxidized thiyl state, resulting in direct regeneration of the reduced enzyme with DTT, or in irreversible inactivation with GTN (E_SH, E_S-, E_S-S, and E_i represent the reduced (thiol/thiolate), oxidized thiyl, oxidized disulfide, and irreversibly inactivated enzyme states; k_ox, k_rev, k_irr, k_red, and k_red′ stand for the rate constants for GTN-induced thiolate oxidation, DTT-reversible disulfide formation, GTN-induced DTT-irreversible inactivation, and DTT-exacted reduction of the E_S-S and E_S· enzyme states, respectively). Note that because of the branching of the reaction after thiol oxidation, irreversible inactivation in the absence of DTT will only be partial, with the extent depending on the concentration of GTN. Inactivation will be slower in the presence of DTT but, on account of the continuous recycling of the disulfide to the active reduced thiol, all of the enzyme should eventually be inactivated irreversibly.