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Glyceraldehyde-3-Phosphate Dehydrogenase Inactivation by Peroxynitrite
ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS
Vol. 360, No. 2, December 15, pp. 187–194, 1998 Article No. BB980932
Glyceraldehyde-3-Phosphate Dehydrogenase Inactivation by Peroxynitrite Jose´ M. Souza and Rafael Radi1 Department of Biochemistry, Facultad de Medicina, Universidad de la Repu´blica, Avenida Gral. Flores 2125, 11800 Montevideo, Uruguay
Received July 2, 1998, and in revised form September 8, 1998
Rabbit muscle glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was inactivated by peroxynitrite under biologically relevant conditions. The decrease of enzymatic activity followed an exponential function, and the concentration of peroxynitrite needed to inactivate 50% of 7 mM GAPDH (IC50) was 17 mM. Hydroxyl radical scavengers did not protect GAPDH from inactivation, but molecules that react directly with peroxynitrite such as cysteine, glutathione, or methionine and the substrate, glyceraldehyde 3-phosphate, afforded significant protection. Assuming simple competition kinetics between scavengers and the enzyme, we estimated a second-order rate constant of (2.5 6 0.5) 3 105 M21 s21 at 25°C and pH 7.4 for the GAPDH tetramer. The loss of enzyme activity was accompanied by protein thiol oxidation (two thiols oxidized per subunit) with only one critical thiol responsible of enzyme inactivation. Indeed, the pH profile of inactivation was consistent with the reaction of GAPDH sulfhydryls (GAPDH-SH) with peroxynitrite. Peroxynitrite-inactivated GAPDH was resistant to arsenite reduction and only 15% recovered by 20 mM dithiothreitol, suggesting that GAPDH-SH has been mainly oxidized to sulfinic or sulfonic acid, with a minor proportion yielding a disulfide. On the other hand, under anaerobic conditions the peroxynitrite precursor, nitric oxide (•NO), only slowly inactivated GAPDH with a rate constant of 11 M21 s21. The remarkable reactivity of the critical thiol group in GAPDH (Cys-149) toward peroxynitrite, which is one order of magnitude higher than that of previously studied sulfhydryls, indicate that it may constitute a preferential intracellular target for peroxynitrite. © 1998 Academic Press Key Words: glyceraldehyde-3-phosphate dehydrogenase; peroxynitrite; nitric oxide; superoxide; free radicals; sulfhydryl oxidation.
1 To whom correspondence should be addressed. Fax: 59829249563. E-mail: [email protected].
0003-9861/98 $25.00 Copyright © 1998 by Academic Press All rights of reproduction in any form reserved.
Glyceraldehyde-3-phosphate dehydrogenase (GAPDH; E.C. 1.2.1.12)2 reversibly catalyzes the oxidation and phosphorylation of D-glyceraldehyde 3-phosphate (GAP). It is a key enzyme in the glycolytic conversion of glucose to pyruvic acid and in the gluconeogenic pathway. The GAPDH catalytic mechanism involves the formation of a hemithioacetal between GAP and the active site thiol followed by oxidation to a thioester which is then phosphorylized to yield the product 1,3-diphosphogliceric acid (1, 2). The cysteine residue of the enzyme involved in the thioester formation was identified as Cys-149 (3), being the most reactive cysteine residue of the enzyme and the target for the selective enzyme inactivation by a number of thiol reagents. Nitric oxide (•NO) is an enzymatically synthesized free radical responsible for a variety of physiological and pathological actions (4, 5). GAPDH has been indicated as an important target for the intracellular effects mediated by •NO. It has been shown that •NO induces a linkage of NAD(H) to a cysteine residue of GAPDH via the transient formation of a S-nitrosothiol intermediate, which results on enzyme inhibition (6–9). GAPDH has been also recognized as an oxidantsensitive enzyme of the glycolytic pathway, since oxidative stress conditions that enhance cellular superoxide (O•2 2 ) and hydrogen peroxide (H2O2) steady-state levels lead to enzyme inactivation (10). In this context, it is important to note that •NO can turn into a powerful oxidizing intermediate upon its fast reaction with O•2 (i.e., peroxynitrite anion, ONOO2; Refs. 11–13) 2 and therefore •NO could mediate GAPDH inactivation by oxidative mechanisms, which are independent of the NAD(H)-dependent covalent modification. In fact, 2 Abbreviations used: GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GAP, D-glyceraldehyde 3-phosphate; SH, thiol; dtpa, diethylenetriaminepentaacetic acid; DTT, dithiothreitol; BSA, bovine serum albumin; DTNB, 5,59-dithiobis(2-nitrobenzoic) acid.
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recent reports have suggested the potential sensitivity of GAPDH toward peroxynitrite (14 –16). Peroxynitrite3 can perform a variety of oxidation and nitration reactions (11–13, 17, 18), but among the most relevant at the cellular level are the reactions with thiol (2SH) groups (12, 19). Low-molecular-weight thiols such as cysteine and glutathione, as well as protein thiols such as the free thiol in bovine serum albumin, have been shown to readily react with peroxynitrite (12, 20). It is remarkable that peroxynitrite usually reacts a thousand times faster with thiols than does H2O2 (12). Peroxynitrite reaction with thiols can lead to a variety of products, including disulfides, and even higher oxidation states of sulfur (e.g., sulfenic, sulfinic, and sulfonic acids). Transient formation of thiyl radicals may also occur through the formation of hydroxyl radical-like intermediates from peroxynitrous acid (ONOOH; Refs. 21, 22) or after formation of the nitrosoperoxocarbonate adduct (ONOOCO2 2 ) following reaction with carbon dioxide (23). Since thiols are critical to the GAPDH activity, we investigated the reactions of peroxynitrite toward GAPDH, with the aim of examining in detail the molecular mechanisms by which peroxynitrite inactivates GAPDH and to reveal the potential contribution of peroxynitrite to •NO-mediated inactivation of cellular GAPDH under oxidative stress conditions. MATERIALS AND METHODS Chemicals. Horse heart cytochrome c (Type III and VI), glutathione (reduced form), methionine, mannitol, dimethyl sulfoxide, diethylenetriaminepentaacetic acid (dtpa), and barium diethylacetal-DLglyceraldehyde 3-phosphate were obtained from Sigma Chemical Co. (St. Louis, MO). Prepacked columns of Sephadex G-25 were from Pharmacia (Piscataway, NJ). Rabbit muscle glyceraldehyde-3-phosphate dehydrogenase was purified by Cori’s method (24) improved by Hill et al. (25). Our preparation was also compared with commercial GAPDH obtained from Boehringer-Mannheim GmbH (Mannheim, Germany). The purity of these different GAPDH preparations was examined by SDS– PAGE electrophoresis (11%), which revealed a main band of around 36 kDa (corresponding to the enzyme monomer) for both preparations. The specific activity from both GAPDH preparations was ;120 mmol NADH/min/mg of protein. Prior to use GAPDH was fully activated in 50 mM sodium pyrophosphate, pH 8.5, 0.1 mM dtpa, and 10 mM dithiothreitol (DTT) for 30 min at 4°C. Excess of thiol was removed using a Sephadex G-25 column equilibrated with the same buffer. GAPDH concentration was determined from A280 values by using e280 5 1.46 3 105 M21 cm21 (26). Peroxynitrite was synthesized in a quenched-flow reactor as described previously (11, 12). The peroxynitrite preparation was treated with manganese dioxide to eliminate excess H2O2. Peroxynitrite concentration was determined spectrophotometrically at 302 nm in 1 M NaOH (e302 5 1670 M21 cm21). Nitric oxide was purchased from AGA Gas Company (Montevideo, Uruguay). Nitric oxide was washed in 5 M KOH and then trapped in the gas-sampling tube containing deoxygenated deionized water. The •NO concentration
was determined electrochemically (Iso-NO sensor; WPI, Sarasota, Florida) or by measuring the oxidation of oxyhemoglobin to methemoglobin according to Ref. (27). Cytochrome c was reduced with excess sodium dithionite immediately before use and purified by gel filtration on Sephadex G-25 using 100 mM potassium phosphate plus 0.1 mM dtpa, pH 7.4, as the elution buffer. The concentration of cytochrome c21 was determined at 550 nm in the same buffer (e550 5 21 3 103 M21 cm21). GAP was obtained as barium diethylacetal-DL-glyceraldehyde 3-phosphate and converted to the free aldehyde and assayed with GAPDH as described by the manufacturer (Sigma). Measurement of GAPDH activity. The assay mixture contained 50 mM Tris–HCl, pH 8.5, 0.1 mM dtpa, 0.25 mM NAD1, 10 mM arsenic acid (sodium salt), 0.5 mM GAP, and 7 mg/ml of enzyme. GAPDH was assayed at 20°C by following the NADH production at 340 nm over the first 30 s after the addition of GAP, using a doublebeam spectrophotometer (Uvikon 930 Kontron spectrophotometer). Exposure of GAPDH to peroxynitrite. Peroxynitrite (0 –100 mM) was added as a single bolus to GAPDH in 0.1 M pyrophosphate, 0.1 mM dtpa, pH 7.4, and incubated for 5 min at 25°C. GAPDH activity was measured immediately after. The influence of the contaminating and decomposition products, nitrite and nitrate, was tested by allowing peroxynitrite first to decompose in buffer before addition to the enzyme (reverse-order addition experiment). The effect of scavengers (cysteine, glutathione, methionine, dimethyl sulfoxide, and mannitol) or enzyme substrates were evaluated by preincubating GAPDH with the studied compound for 2 min before the addition of peroxynitrite. Control experiments to rule out direct effects of the scavengers on GAPDH activity were also performed. For experiments to study the pH profile of inactivation GAPDH was preincubated for 5 min at different pH levels ranging from 6.0 to 9.3 and then exposed to peroxynitrite. The final pH of the reactions was always measured. Aliquots were withdrawn and GAPDH activity was assayed. Competition experiments. Simple competition kinetics experiments were performed in order to estimate a second-order rate constant of peroxynitrite with GAPDH, as previously (28, 29). Molecules that react with peroxynitrite with known second-order rate constants such as glutathione, cysteine, and cytochrome c21 were used. The fraction of protection from GAPDH inactivation, Fi, was calculated as elsewhere (30). Titration of sulfhydryl groups. The thiol groups of GAPDH were titrated in 50 mM Tris–HCl, pH 8, 5 mM EDTA, 1% SDS, and 1 mM 5,59-dithiobis(2-nitrobenzoic) acid (DTNB). A molar extinction coefficient of 1.36 3 104 M21 cm21 for the anion of thionitrobenzoic acid was used (12, 31). Exposure to GAPDH to •NO. GAPDH was incubated in 0.1 M Tris–HCl, 0.1 mM dtpa, pH 7.2, under aerobic or anaerobic conditions. Anaerobic conditions were accomplished by purging the reaction mixture with argon for 10 min. Then, authentic •NO was added through the rubber septum of a tightly sealed tube using a gas-tight Hamilton syringe. Aliquots were withdrawn at different times to assay the enzyme activity. Reductive recovery of inactivated GAPDH. GAPDH was exposed to peroxynitrite, •NO, or iodosobenzoate in 0.1 M pyrophosphate, 0.1 mM dtpa, pH 7.4. Then, the enzyme was incubated with 14 mM sodium arsenite or 20 mM DTT for 30 min at 25°C. Arsenite was used to reduce sulfenic acid derivatives back to thiols, as described previously (12). The percentage of GAPDH recovered after inactivation was calculated as in previous work (12, 28).
RESULTS 3
The term peroxynitrite is used to refer the sum of both peroxynitrite anion (ONOO2) and peroxynitrous acid (ONOOH).
Exposure of GAPDH to peroxynitrite showed a dosedependent inactivation (Fig. 1). The decay of enzymatic
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GAPDH INACTIVATION BY PEROXYNITRITE TABLE I
Inhibition of Peroxynitrite-Mediated Inactivation of GAPDH by Different Scavengers Condition
% Initial activity
Control 1ONOO2 1Dimethyl sulfoxide (25 mM) 1Mannitol (25 mM) 1DTT (10 mM) 1Methionine (10 mM) 1Cysteine (5 mM) 1Bicarbonate (25 mM)
100 6 2 761 261 862 105 6 3 102 6 4 108 6 5 22 6 2
Note. GAPDH (6 mM) was incubated with scavengers in 0.1 M pyrophosphate, 0.1 mM dtpa, pH 7.4, for 10 min and then exposed to 40 mM peroxynitrite. Aliquots were withdrawn and GAPDH activity was assayed.
FIG. 1. Inactivation of GAPDH by peroxynitrite. GAPDH (0.6 (F), 2.9 (ƒ), 6.9 (Œ), and 17.3 (h) mM) was incubated with peroxynitrite in 0.1 M pyrophosphate, 0.1 mM dtpa, pH 7.4, at 25°C for 5 min. Aliquots were withdrawn, and GAPDH activity was assayed. The reverse-order addition is also shown, corresponding to 6.9 mM of GAPDH (■).
activity followed an exponential function and the concentration of peroxynitrite needed to inactivate 50% of the initial activity (IC50) was calculated at different initial GAPDH concentrations. IC50 ranged from 3.2 to 33 mM of peroxynitrite for 0.6 to 17.3 mM of enzyme, respectively. A similar behavior was previously shown for peroxynitrite-mediated inactivation of aconitase and alcohol dehydrogenase (28, 32). At the high GAPDH concentration (i.e., 17.3 mM), it can be estimated that about four molecules of peroxynitrite (i.e., IC50 5 33 mM) were required for inactivation of one molecule of tetrameric enzyme (i.e., one peroxynitrite per enzyme monomer). Reverse-order addition of peroxynitrite to GAPDH did not result in enzyme inactivation. In order to explore the mechanism by which peroxynitrite inactivates GAPDH, several molecules that react with either peroxynitrite or secondary oxidants with hydroxyl radical-like reactivity from peroxynitrite decomposition were evaluated. Table I shows that hydroxyl radical scavengers such as mannitol or dimethyl sulfoxide did not protect GAPDH from inactivation, while molecules that react directly with peroxynitrite (cysteine, methionine, and DTT) afforded complete protection. Also, the effect of the bicarbonate– carbon dioxide pair was evaluated, as it has been recently shown that ONOO2 reaction with CO2 is a highly relevant reaction in biological systems (33); 25 mM added bicarbonate, in equilibrium with ;1.2 mM CO2 resulted in a rather modest 16% protection from inactivation.
Using simple competition kinetics (28 –30) for the protection of GAPDH by different concentrations of glutathione and cysteine (k 5 740 and 2500 M21 s21 at pH 7.4 and 25°C, respectively (12, 20)), it can be estimated that the rate constant of peroxynitrite reaction with the GAPDH tetramer is (2.5 6 0.2) 3 105 M21 s21 (Fig. 2).
FIG. 2. Rate constant between peroxynitrite and GAPDH. GAPDH (3.4 mM) was preincubated for 10 min with different concentrations of GSH (■) or cysteine (h) in 0.1 M pyrophosphate, 0.1 mM dtpa, pH 7.4, at 25°C and then exposed to 20 mM peroxynitrite. Aliquots were withdrawn, and GAPDH activity was assayed. Fi represents the fraction of protection of GAPDH in the presence of the scavenger ([S]); ks, the rate constant for the reaction of ONOO2 with the scavenger; and kd, the rate constant for the reaction of peroxynitrite with GAPDH. Plotting Fi[GAPDH]/(1 2 Fi)ks against [S] results in a straight line with the slope 1/kd, which allows the determination of kd (28, 30).
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Competition Experiments between GAPDH and Cytochrome c21 Cyt c21
GAPDH
Control 1ONOO2 1Competitor
Activity (mmol/min/mg)
% Initial activity
Concentration (mM)
% Initial concentration
120 22.0 45.2
100 18.3 37.7
80 58.6 73.8
100 73.2 92.3
Note. GAPDH (17 mM) and cytochrome c21 (80 mM) were exposed to 50 mM peroxynitrite in 0.1 M pyrophosphate, 0.1 mM dtpa, pH 7.4, for 10 min at 25°C in the absence or presence (1Competitor) of the other target molecule. Then, aliquots of the reaction mixtures were assayed by measuring the absorbance at 550 nm and GAPDH activity. The fraction of protection (Fi) for each target was determined and the rate constant for the GAPDH-peroxynitrite was determined independently in both assays as described previously (28, 29).
To further confirm the estimated rate constant between GAPDH and peroxynitrite obtained in Fig. 2, we performed a competition experiment between GAPDH and cytochrome c21 against peroxynitrite. Cytochrome c21 is oxidized to cytochrome c31 by peroxynitrite with a second-order rate constant of 1.32 3 104 M21 s21 at pH 7.4 and 25°C (34). The oxidation of cytochrome c21 by peroxynitrite was measured with and without GAPDH and, similarly, in the same experiment GAPDH inactivation by peroxynitrite was measured in the presence and absence of cytochrome c21 (Table II). From the data in Table II and using independent cal-
culations for either the fraction of protection of GAPDH or that of cytochrome c21, a rate constant of 2.0 3 105 M21 s21 was estimated, in good agreement with the data in Fig. 2. We evaluated whether the presence of substrate would protect the GAPDH active site from peroxynitrite-mediated inactivation. Glyceraldehyde 3-phosphate was able to protect GAPDH in a dose-dependent manner as shown in Fig. 3, reaching 100% protection at infinite substrate concentration (Fig. 3, inset). The other substrates, NAD1 and arsenate or phosphate, did not protect GAPDH (data not shown). It therefore seems probable that peroxyni-
FIG. 3. Peroxynitrite-mediated inactivation in the presence of substrate. GAPDH (0.52 mM) was incubated with different concentrations of glyceraldehyde 3-phosphate in 0.1 M pyrophosphate, 0.1 mM dtpa, pH 7.4, at 25°C for 5 min and then exposed to 30 mM peroxynitrite. (Inset) A Scachartd-like plot of the data.
GAPDH INACTIVATION BY PEROXYNITRITE
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FIG. 4. Influence of pH on peroxynitrite-mediated inactivation. GAPDH (1.4 mM) was incubated for 5 min in 0.1 M pyrophosphate, 0.1 mM dtpa at different pH levels and then exposed to 10 mM peroxynitrite. Aliquots were withdrawn, and GAPDH activity was assayed. Conditions were control (1), peroxynitrite (■), and 5 mM methionine (h).
trite and GAP interact with GAPDH at identical or adjacent sites on the enzyme. The inactivation of GAPDH by peroxynitrite was strongly pH-dependent (Fig. 4), with maximal inactivation obtained at alkaline pH. However, a significant fraction of the enzyme (;15%) was still inactivated at acidic pH. Methionine was able to protect GAPDH from inactivation mostly at acidic pH, in agreement with its reaction with peroxynitrous acid (35). During peroxynitrite-mediated GAPDH inactivation there was enzyme thiol oxidation (Fig. 5). While each monomer contains up to four thiols (1) (Fig. 5), only up to two thiols per enzyme monomer disappeared in the peroxynitrite concentration range of 0 – 40 mM, with only one critical thiol responsible for .90% enzyme inactivation as shown by the Tsou plot (36), where remaining enzyme activity was plotted against sulfhydryl loss (Fig. 5, inset). Arsenite and dithiothreitolmediated reduction of oxidized thiols permitted determination of the reversibility of sulfhydryl oxidation and aided identification of thiol oxidation states after peroxynitrite treatment (12, 37). Arsenite reduces sulfenic acid to sulfhydryl but does not reduce disulfides (37), while DTT reduces disulfides to thiols. Peroxynitrite-mediated oxidation of GAPDH thiols resulted in a product resistant to arsenite and only 15% DTT-recoverable (Table III). In contrast, iodosobenzo-
ate-mediated GAPDH thiol oxidation was 71% reduced by arsenite, suggesting a significant formation of sulfenic acid with this reagent as was previously shown by Parker and Allison (38). For comparative purposes with peroxynitrite reactivity, exposure of GAPDH to authentic •NO anaerobically (Fig. 6) resulted in a slow decrease in enzyme activity. Fitting this data to an exponential function, a second-order rate constant between •NO and GAPDH of 11.2 6 0.5 M21 s21 at pH 7.2 at 25°C is estimated. Preincubation of GAPDH with DTT completely protected against •NO-mediated inactivation. Postincubation with either DTT or arsenite resulted in some recovery of enzymic activity (Table III). On the other hand, under aerobic conditions where authentic •NO evolves to dinitrogen trioxide (N2O3), GAPDH was rapidly inactivated (in less than 30 s) to 60% of initial activity (Table III), with preincubation with GAP or DTT being completely protective. The addition of DTT to aerobic •NO-inactivated GAPDH caused a total reactivation of the enzyme (Table III). DISCUSSION
In this study we demonstrate that GAPDH is highly sensitive to inactivation by peroxynitrite. The high sensitivity is due to the high second-order rate con-
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FIG. 5. Sulfhydryl oxidation during GAPDH inactivation by peroxynitrite. GAPDH (2 mM) was incubated for 5 min in 0.1 M pyrophosphate, 0.1 mM dtpa, pH 7.4, at 25°C with different concentrations of peroxynitrite. Then aliquots were withdrawn, and GAPDH activity was assayed (■). Sulfhydryls were quantitated using the DTNB reaction in the presence of 1% SDS (h). (Inset) Fractional activity against oxidized sulfydryls per enzyme monomer.
stant of (2.5 6 0.5) 3 105 M21 s21 obtained for the GAPDH–peroxynitrite reaction and the 4:1 (peroxynitrite:GAPDH tetramer ratio) inactivation stoichiometry. The sensitivity of this enzyme compares well with that of others having oxidant-sensitive active sites, including alcohol dehydrogenase (31), aconitase (28), and glutathione peroxidase (39). In line with this concept, it has been recently shown that in Escherichia coli, the family of [4Fe-4S] cluster-containing dehydratases (aconitase, 6-phosphogluconate dehydratase, and fumarase A) and GAPDH were primary targets of peroxynitrite-mediated inactivation of enzymes (15). Peroxynitrite-mediated inactivation of GAPDH involving interactions at the active site can be concluded by the total protection afforded by excess substrate (Fig. 3). Substrate binding involves thioester bond formation between GAP and the essential SH groups of the enzyme, thus precluding peroxynitrite attack on the thiol. Indeed, inactivation of GAPDH by peroxynitrite consists of the oxidation of sulfhydryl groups of the enzyme (Fig. 5), one of which is responsible of most of the enzyme activity loss.
It is worth noting that the reaction of peroxynitrite with each subunit of GAPDH, which should primarily rely on interactions with a single critical thiol (Fig. 5), is one order of magnitude faster (6 3 104 M21 s21 per monomer) than that with cysteine, glutathione, and the single thiol group of bovine serum albumin (BSA) (12, 20, 22). This high rate constant between peroxynitrite and GAPDH may be dependent on the strongly hydrophilic and positively charged environment of the enzyme active site which would favor attraction of peroxynitrite toward the mercaptide (1, 40). Interestingly, one critical thiol group of GAPDH (Cys149) has been also shown to react with H2O2 with rates more than a hundred times faster than with the single thiol group of BSA at pH 7.0 (.100 M21 s21 (40) versus ;1 M21 s21 (12), respectively), confirming the unique reactivity of this residue. The preferential direct reaction of peroxynitrite with the thiol group of the enzyme is further supported by the protection afforded by cysteine, DTT, and methionine (and not by hydroxyl radical scavengers) and the pH profile of inactivation (12) (Table I and Fig. 4).
GAPDH INACTIVATION BY PEROXYNITRITE TABLE III
Reductive Recovery of Peroxynitrite, Nitric Oxide, and Iodosobenzoate-Oxidized GAPDH Condition
Reductant
% Initial activity
% Recovery
1ONOO2 1ONOO2 1ONOO2 1•NO (anaerobic) 1•NO (anaerobic) 1•NO (anaerobic) 1•NO (aerobic) 1•NO (aerobic) 1Iodosobenzoate 1Iodosobenzoate
— 1Arsenite 1DTT — 1Arsenite 1DTT — 1DTT — 1Arsenite
3.1 6 0.2 3.5 6 0.3 18 6 1 12 6 1 36 6 1 43 6 2 60 6 2 105 6 3 6.5 6 0.3 73 6 1
— 0.4 15 — 27 35 — 100 — 71
Note. GAPDH (2.7 mM) was exposed to 40 mM peroxynitrite, 200 mM •NO or 27 mM iodosobenzoate in 0.1 M pyrophosphate, 0.1 mM dtpa, pH 7.4, for 10 min (peroxynitrite and iodosobenzoate) or 30 min (•NO) at 25°C. Then, incubations with sodium arsenite or DTT were performed as indicated under Materials and Methods. Samples were immediately assayed for GAPDH activity. The percentage of recovery was calculated.
Although maximal inactivation was obtained at alkaline pH, some GAPDH inactivation was seen at acidic pH (Fig. 4), which is most likely dependent on the formation of secondary products from peroxynitrous acid (11, 20), which can oxidize sulfhydryls via a oneelectron oxidation mechanism (22). This reaction predominantly occurs with the thiolate form of the sulfhydryl, which in the case of GAPDH may be relevant even at acidic pH (i.e., 6.0) due to the low pKa of the critical thiol group of about 7.5 (40). This secondary mechanism can also explain recent results on the detection of thiyl radical in apo-GAPDH treated with 1,3-morpholinosydnonimine and peroxynitrite (16). The fast reaction between ONOO2 and CO2 (k 5 5.8 3 104 M21 s21 at 37°C; Ref. 33) leads to the formation of the nitrosoperoxocarbonate adduct, ONOOCO2 2, and this reaction would largely outcompete (;98%) the reaction of peroxynitrite with GAPDH under the conditions of Table I. However, protection from inactivation was marginal, which is consistent with the reported reactivity of ONOOCO2 2 toward thiols (33), causing enzyme inactivation in its own right. Although there is loss of two thiols per enzyme monomer, disulfide does not appear to be the main oxidation product as judged by the lack of reactivation by DTT (Table III). A minor amount of disulfide bond formation between Cys-149 and nearby Cys-153 may occur, although these residues can have only limited interactions as the side chain of Tyr-311 lies between the –SH groups of these two cysteines, inhibiting disulfide formation (41). Thus, considering that Cys-149 may be relatively isolated, it may have been oxidized by peroxynitrite to –SOH (sulfenic acid), –SO2H (sulfinic acid), or –SO3H (sulfonic acid) or to a combination of
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these. The mild oxidizing agents iodosobenzoate (Table III) or low concentrations of hydrogen peroxide (40) caused SH oxidation to sulfenic acid, which could be recovered by sodium arsenite. On the contrary, peroxynitrite oxidized the essential thiol of GAPDH toward higher oxidation states, sulfinic or sulfonic acid, because sodium arsenite was unable to recover activity from peroxynitrite-mediated GAPDH inactivation. This finding is similar to that with the single thiol group of BSA, where H2O2 oxidized it to an arsenitereducible product, while peroxynitrite oxidized it to a arsenite-resistant product (12). The mechanisms by which peroxynitrite promote formation of sulfinic and/or sulfonic acid derivatives in isolated protein thiols are not known at present. GAPDH has been implicated as one of the major intracellular targets of •NO, and the loss of GAPDH activity has been attributed to the linkage of NAD(H) to a cysteine residue of the active site. Herein, we have shown a slow inactivation process by authentic •NO on GAPDH under anaerobic conditions and in the absence of metals or NAD(H) (Fig. 6). This agrees with a slow nitric oxide-mediated oxidation of thiols under anaerobic conditions which may involve the formation of sulfenic acid derivatives (Table III), as previously reported (42). Aerobically, there was a more rapid •NOmediated inactivation, which would primarily depend on the formation of dinitrogen trioxide (N2O3), which
FIG. 6. Effect of •NO on GAPDH activity. GAPDH (1.8 mM) was incubated anaerobically with none (1), 200 mM •NO (Œ), and 200 mM • NO (h) in the presence of 1 mM DTT in 0.1 M Tris–HCl, 0.1 mM dtpa, pH 7.2, at 25°C, and activity was assayed in aliquots at different time points.
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promotes nitrosation of Cys-149 and loss of enzyme activity. Our study shows that GAPDH is highly reactive toward peroxynitrite, with this reactivity mostly dependent on the rather unique properties of the critical thiol group of the enzyme, Cys-149. GAPDH is an enzyme which is abundant in various tissues. For instance, in skeletal muscle, up to 10% of soluble protein is GAPDH and significant amounts also exist in heart muscle, brain, kidney, and liver (1). Thus, the ubiquity of GAPDH and its significant rate constant imply that GAPDH inactivation by peroxynitrite might be a relevant mechanism which may alter cellular glycolysis and/or gluconeogenesis as well as other functions recently ascribed to the enzyme (43). This process may occur even in the presence of biological levels of CO2, both because the reaction of cellular GAPDH-SH may compete well with the formation of ONOOCO2 2 , but also because ONOOCO2 is also able to efficiently oxi2 dize thiols. ACKNOWLEDGMENTS We thank Dr. Gerardo Ferrer for helpful comments and critical reading of the manuscript. This work was supported by grants from CSIC, Uruguay, to J.M.S. and CONICYT, Uruguay, and SAREC, Sweden, to R.R.
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14. Mohr, S., Stamler, J. S., and Bru¨ne, B. (1994) FEBS Lett. 348, 223–227. 15. Keyer, K., and Imlay, J. A. (1997) J. Biol. Chem. 272, 27652– 27659. 16. Minetti, M., Pietraforte, D., Di Stasi, A. M. M., and Mallozzi, C. (1996) Biochem. J. 319, 369 –375. 17. Ischiropoulos, H., Zhu, L., Chen, J., Tsai, M., Martin, J. C., Smith, C. D., and Beckman, J. S. (1992) Arch. Biochem. Biophys. 298, 431– 437. 18. Beckman, J. S., Ischiropoulos, H., Zhu, L., van der Woerd, M., Smith, C. D., Chen, J., Harrison, J., Martin, J. C., and Tsai, M. (1992) Arch. Biochem. Biophys. 298, 438 – 455. 19. Augusto, O., and Radi, R. (1995) in Biothiols (Cadenas, E., and Packer, L., Eds.), pp. 83–116, Dekker, New York. 20. Koppenol, W. H., Pryor, W. A., Moreno, J. J., Ischiropoulos, H., and Beckman, J. S. (1992) Chem. Res. Toxicol. 5, 834 – 842. 21. Ohara, A., Gatti, R., and Radi, R. (1994). Arch. Biochem. Biophys. 310, 118 –125. 22. Quijano, C., Alvarez, B., Gatti, R., Augusto, O., and Radi, R. (1997) Biochem. J. 322, 167–173. 23. Scorza, G., and Minetti, M. (1998) Biochem. J. 329, 405– 413. 24. Cori, G. T., Slein, M. W., and Cori, C. F. (1948) J. Biol. Chem. 174, 605– 618. 25. Hill, E. J., Meriwether, B. P., and Park, J. H. (1975) Anal. Biochem. 63, 175–182. 26. Fox, J. B., and Dandliker, W. B. (1956) J. Biol. Chem. 221, 1005–1017. 27. Winternbourn, C. C. (1990) Methods Enzymol. 186, 265–272. 28. Castro, L., Rodrı´guez, M., and Radi, R. (1994) J. Biol. Chem. 269, 29409 –29415. 29. Radi, R. (1996) Methods Enzymol. 268, 354 –366. 30. Winternbourn, C. C. (1987) Free Radical Biol. Med. 3, 33–39. 31. Ellman, G. L. (1959) Arch. Biochem. Biophys. 82, 70 –77. 32. Crow, J., Beckman, J. S., and McCord, J. M. (1995) Biochemistry 34, 3544 –3552. 33. Radi, R., Denicola, A., and Freeman, B. A. (1998) Methods Enzymol. 301, 353–367. 34. Thomson, L., Trujillo, M., Telleri, R., and Radi, R. (1995) Arch. Biochem. Biophys. 319, 491– 497. 35. Pryor, W. A., Xin, X., and Squadrito, G. L. (1994) Proc. Natl. Acad. Sci. USA 91, 11173–11177. 36. Tsou, C. L. (1962) Sci. Sin. 11, 1535–1558. 37. Torchinsky, Y. M. (1981) in Sulfur in Proteins, pp. 48 –57, Pergamon, New York. 38. Parker, D. J., and Allison, W. S. (1969) J. Biol. Chem. 244, 180 –189. 39. Padmaja, S., Squadrito, G. L., and Pryor, W. A. (1998) Arch. Biochem. Biophys. 349, 1– 6. 40. Little, C., and O’Brien (1969) Eur. J. Biochem. 10, 533–538. 41. Allison, W. S. (1976) Acc. Chem. Res. 9, 293–299. 42. DeMaster, E. G., Quast, B. J., Redfern, B., and Nagasawa, H. T. (1995) Biochemistry 34, 11494 –11499. 43. Bru¨ne, B., and Lapetina, E. G. (1996) Methods Enzymol. 269, 400 – 407.
Vol. 360, No. 2, December 15, pp. 187–194, 1998 Article No. BB980932
Glyceraldehyde-3-Phosphate Dehydrogenase Inactivation by Peroxynitrite Jose´ M. Souza and Rafael Radi1 Department of Biochemistry, Facultad de Medicina, Universidad de la Repu´blica, Avenida Gral. Flores 2125, 11800 Montevideo, Uruguay
Received July 2, 1998, and in revised form September 8, 1998
Rabbit muscle glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was inactivated by peroxynitrite under biologically relevant conditions. The decrease of enzymatic activity followed an exponential function, and the concentration of peroxynitrite needed to inactivate 50% of 7 mM GAPDH (IC50) was 17 mM. Hydroxyl radical scavengers did not protect GAPDH from inactivation, but molecules that react directly with peroxynitrite such as cysteine, glutathione, or methionine and the substrate, glyceraldehyde 3-phosphate, afforded significant protection. Assuming simple competition kinetics between scavengers and the enzyme, we estimated a second-order rate constant of (2.5 6 0.5) 3 105 M21 s21 at 25°C and pH 7.4 for the GAPDH tetramer. The loss of enzyme activity was accompanied by protein thiol oxidation (two thiols oxidized per subunit) with only one critical thiol responsible of enzyme inactivation. Indeed, the pH profile of inactivation was consistent with the reaction of GAPDH sulfhydryls (GAPDH-SH) with peroxynitrite. Peroxynitrite-inactivated GAPDH was resistant to arsenite reduction and only 15% recovered by 20 mM dithiothreitol, suggesting that GAPDH-SH has been mainly oxidized to sulfinic or sulfonic acid, with a minor proportion yielding a disulfide. On the other hand, under anaerobic conditions the peroxynitrite precursor, nitric oxide (•NO), only slowly inactivated GAPDH with a rate constant of 11 M21 s21. The remarkable reactivity of the critical thiol group in GAPDH (Cys-149) toward peroxynitrite, which is one order of magnitude higher than that of previously studied sulfhydryls, indicate that it may constitute a preferential intracellular target for peroxynitrite. © 1998 Academic Press Key Words: glyceraldehyde-3-phosphate dehydrogenase; peroxynitrite; nitric oxide; superoxide; free radicals; sulfhydryl oxidation.
1 To whom correspondence should be addressed. Fax: 59829249563. E-mail: [email protected].
0003-9861/98 $25.00 Copyright © 1998 by Academic Press All rights of reproduction in any form reserved.
Glyceraldehyde-3-phosphate dehydrogenase (GAPDH; E.C. 1.2.1.12)2 reversibly catalyzes the oxidation and phosphorylation of D-glyceraldehyde 3-phosphate (GAP). It is a key enzyme in the glycolytic conversion of glucose to pyruvic acid and in the gluconeogenic pathway. The GAPDH catalytic mechanism involves the formation of a hemithioacetal between GAP and the active site thiol followed by oxidation to a thioester which is then phosphorylized to yield the product 1,3-diphosphogliceric acid (1, 2). The cysteine residue of the enzyme involved in the thioester formation was identified as Cys-149 (3), being the most reactive cysteine residue of the enzyme and the target for the selective enzyme inactivation by a number of thiol reagents. Nitric oxide (•NO) is an enzymatically synthesized free radical responsible for a variety of physiological and pathological actions (4, 5). GAPDH has been indicated as an important target for the intracellular effects mediated by •NO. It has been shown that •NO induces a linkage of NAD(H) to a cysteine residue of GAPDH via the transient formation of a S-nitrosothiol intermediate, which results on enzyme inhibition (6–9). GAPDH has been also recognized as an oxidantsensitive enzyme of the glycolytic pathway, since oxidative stress conditions that enhance cellular superoxide (O•2 2 ) and hydrogen peroxide (H2O2) steady-state levels lead to enzyme inactivation (10). In this context, it is important to note that •NO can turn into a powerful oxidizing intermediate upon its fast reaction with O•2 (i.e., peroxynitrite anion, ONOO2; Refs. 11–13) 2 and therefore •NO could mediate GAPDH inactivation by oxidative mechanisms, which are independent of the NAD(H)-dependent covalent modification. In fact, 2 Abbreviations used: GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GAP, D-glyceraldehyde 3-phosphate; SH, thiol; dtpa, diethylenetriaminepentaacetic acid; DTT, dithiothreitol; BSA, bovine serum albumin; DTNB, 5,59-dithiobis(2-nitrobenzoic) acid.
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recent reports have suggested the potential sensitivity of GAPDH toward peroxynitrite (14 –16). Peroxynitrite3 can perform a variety of oxidation and nitration reactions (11–13, 17, 18), but among the most relevant at the cellular level are the reactions with thiol (2SH) groups (12, 19). Low-molecular-weight thiols such as cysteine and glutathione, as well as protein thiols such as the free thiol in bovine serum albumin, have been shown to readily react with peroxynitrite (12, 20). It is remarkable that peroxynitrite usually reacts a thousand times faster with thiols than does H2O2 (12). Peroxynitrite reaction with thiols can lead to a variety of products, including disulfides, and even higher oxidation states of sulfur (e.g., sulfenic, sulfinic, and sulfonic acids). Transient formation of thiyl radicals may also occur through the formation of hydroxyl radical-like intermediates from peroxynitrous acid (ONOOH; Refs. 21, 22) or after formation of the nitrosoperoxocarbonate adduct (ONOOCO2 2 ) following reaction with carbon dioxide (23). Since thiols are critical to the GAPDH activity, we investigated the reactions of peroxynitrite toward GAPDH, with the aim of examining in detail the molecular mechanisms by which peroxynitrite inactivates GAPDH and to reveal the potential contribution of peroxynitrite to •NO-mediated inactivation of cellular GAPDH under oxidative stress conditions. MATERIALS AND METHODS Chemicals. Horse heart cytochrome c (Type III and VI), glutathione (reduced form), methionine, mannitol, dimethyl sulfoxide, diethylenetriaminepentaacetic acid (dtpa), and barium diethylacetal-DLglyceraldehyde 3-phosphate were obtained from Sigma Chemical Co. (St. Louis, MO). Prepacked columns of Sephadex G-25 were from Pharmacia (Piscataway, NJ). Rabbit muscle glyceraldehyde-3-phosphate dehydrogenase was purified by Cori’s method (24) improved by Hill et al. (25). Our preparation was also compared with commercial GAPDH obtained from Boehringer-Mannheim GmbH (Mannheim, Germany). The purity of these different GAPDH preparations was examined by SDS– PAGE electrophoresis (11%), which revealed a main band of around 36 kDa (corresponding to the enzyme monomer) for both preparations. The specific activity from both GAPDH preparations was ;120 mmol NADH/min/mg of protein. Prior to use GAPDH was fully activated in 50 mM sodium pyrophosphate, pH 8.5, 0.1 mM dtpa, and 10 mM dithiothreitol (DTT) for 30 min at 4°C. Excess of thiol was removed using a Sephadex G-25 column equilibrated with the same buffer. GAPDH concentration was determined from A280 values by using e280 5 1.46 3 105 M21 cm21 (26). Peroxynitrite was synthesized in a quenched-flow reactor as described previously (11, 12). The peroxynitrite preparation was treated with manganese dioxide to eliminate excess H2O2. Peroxynitrite concentration was determined spectrophotometrically at 302 nm in 1 M NaOH (e302 5 1670 M21 cm21). Nitric oxide was purchased from AGA Gas Company (Montevideo, Uruguay). Nitric oxide was washed in 5 M KOH and then trapped in the gas-sampling tube containing deoxygenated deionized water. The •NO concentration
was determined electrochemically (Iso-NO sensor; WPI, Sarasota, Florida) or by measuring the oxidation of oxyhemoglobin to methemoglobin according to Ref. (27). Cytochrome c was reduced with excess sodium dithionite immediately before use and purified by gel filtration on Sephadex G-25 using 100 mM potassium phosphate plus 0.1 mM dtpa, pH 7.4, as the elution buffer. The concentration of cytochrome c21 was determined at 550 nm in the same buffer (e550 5 21 3 103 M21 cm21). GAP was obtained as barium diethylacetal-DL-glyceraldehyde 3-phosphate and converted to the free aldehyde and assayed with GAPDH as described by the manufacturer (Sigma). Measurement of GAPDH activity. The assay mixture contained 50 mM Tris–HCl, pH 8.5, 0.1 mM dtpa, 0.25 mM NAD1, 10 mM arsenic acid (sodium salt), 0.5 mM GAP, and 7 mg/ml of enzyme. GAPDH was assayed at 20°C by following the NADH production at 340 nm over the first 30 s after the addition of GAP, using a doublebeam spectrophotometer (Uvikon 930 Kontron spectrophotometer). Exposure of GAPDH to peroxynitrite. Peroxynitrite (0 –100 mM) was added as a single bolus to GAPDH in 0.1 M pyrophosphate, 0.1 mM dtpa, pH 7.4, and incubated for 5 min at 25°C. GAPDH activity was measured immediately after. The influence of the contaminating and decomposition products, nitrite and nitrate, was tested by allowing peroxynitrite first to decompose in buffer before addition to the enzyme (reverse-order addition experiment). The effect of scavengers (cysteine, glutathione, methionine, dimethyl sulfoxide, and mannitol) or enzyme substrates were evaluated by preincubating GAPDH with the studied compound for 2 min before the addition of peroxynitrite. Control experiments to rule out direct effects of the scavengers on GAPDH activity were also performed. For experiments to study the pH profile of inactivation GAPDH was preincubated for 5 min at different pH levels ranging from 6.0 to 9.3 and then exposed to peroxynitrite. The final pH of the reactions was always measured. Aliquots were withdrawn and GAPDH activity was assayed. Competition experiments. Simple competition kinetics experiments were performed in order to estimate a second-order rate constant of peroxynitrite with GAPDH, as previously (28, 29). Molecules that react with peroxynitrite with known second-order rate constants such as glutathione, cysteine, and cytochrome c21 were used. The fraction of protection from GAPDH inactivation, Fi, was calculated as elsewhere (30). Titration of sulfhydryl groups. The thiol groups of GAPDH were titrated in 50 mM Tris–HCl, pH 8, 5 mM EDTA, 1% SDS, and 1 mM 5,59-dithiobis(2-nitrobenzoic) acid (DTNB). A molar extinction coefficient of 1.36 3 104 M21 cm21 for the anion of thionitrobenzoic acid was used (12, 31). Exposure to GAPDH to •NO. GAPDH was incubated in 0.1 M Tris–HCl, 0.1 mM dtpa, pH 7.2, under aerobic or anaerobic conditions. Anaerobic conditions were accomplished by purging the reaction mixture with argon for 10 min. Then, authentic •NO was added through the rubber septum of a tightly sealed tube using a gas-tight Hamilton syringe. Aliquots were withdrawn at different times to assay the enzyme activity. Reductive recovery of inactivated GAPDH. GAPDH was exposed to peroxynitrite, •NO, or iodosobenzoate in 0.1 M pyrophosphate, 0.1 mM dtpa, pH 7.4. Then, the enzyme was incubated with 14 mM sodium arsenite or 20 mM DTT for 30 min at 25°C. Arsenite was used to reduce sulfenic acid derivatives back to thiols, as described previously (12). The percentage of GAPDH recovered after inactivation was calculated as in previous work (12, 28).
RESULTS 3
The term peroxynitrite is used to refer the sum of both peroxynitrite anion (ONOO2) and peroxynitrous acid (ONOOH).
Exposure of GAPDH to peroxynitrite showed a dosedependent inactivation (Fig. 1). The decay of enzymatic
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GAPDH INACTIVATION BY PEROXYNITRITE TABLE I
Inhibition of Peroxynitrite-Mediated Inactivation of GAPDH by Different Scavengers Condition
% Initial activity
Control 1ONOO2 1Dimethyl sulfoxide (25 mM) 1Mannitol (25 mM) 1DTT (10 mM) 1Methionine (10 mM) 1Cysteine (5 mM) 1Bicarbonate (25 mM)
100 6 2 761 261 862 105 6 3 102 6 4 108 6 5 22 6 2
Note. GAPDH (6 mM) was incubated with scavengers in 0.1 M pyrophosphate, 0.1 mM dtpa, pH 7.4, for 10 min and then exposed to 40 mM peroxynitrite. Aliquots were withdrawn and GAPDH activity was assayed.
FIG. 1. Inactivation of GAPDH by peroxynitrite. GAPDH (0.6 (F), 2.9 (ƒ), 6.9 (Œ), and 17.3 (h) mM) was incubated with peroxynitrite in 0.1 M pyrophosphate, 0.1 mM dtpa, pH 7.4, at 25°C for 5 min. Aliquots were withdrawn, and GAPDH activity was assayed. The reverse-order addition is also shown, corresponding to 6.9 mM of GAPDH (■).
activity followed an exponential function and the concentration of peroxynitrite needed to inactivate 50% of the initial activity (IC50) was calculated at different initial GAPDH concentrations. IC50 ranged from 3.2 to 33 mM of peroxynitrite for 0.6 to 17.3 mM of enzyme, respectively. A similar behavior was previously shown for peroxynitrite-mediated inactivation of aconitase and alcohol dehydrogenase (28, 32). At the high GAPDH concentration (i.e., 17.3 mM), it can be estimated that about four molecules of peroxynitrite (i.e., IC50 5 33 mM) were required for inactivation of one molecule of tetrameric enzyme (i.e., one peroxynitrite per enzyme monomer). Reverse-order addition of peroxynitrite to GAPDH did not result in enzyme inactivation. In order to explore the mechanism by which peroxynitrite inactivates GAPDH, several molecules that react with either peroxynitrite or secondary oxidants with hydroxyl radical-like reactivity from peroxynitrite decomposition were evaluated. Table I shows that hydroxyl radical scavengers such as mannitol or dimethyl sulfoxide did not protect GAPDH from inactivation, while molecules that react directly with peroxynitrite (cysteine, methionine, and DTT) afforded complete protection. Also, the effect of the bicarbonate– carbon dioxide pair was evaluated, as it has been recently shown that ONOO2 reaction with CO2 is a highly relevant reaction in biological systems (33); 25 mM added bicarbonate, in equilibrium with ;1.2 mM CO2 resulted in a rather modest 16% protection from inactivation.
Using simple competition kinetics (28 –30) for the protection of GAPDH by different concentrations of glutathione and cysteine (k 5 740 and 2500 M21 s21 at pH 7.4 and 25°C, respectively (12, 20)), it can be estimated that the rate constant of peroxynitrite reaction with the GAPDH tetramer is (2.5 6 0.2) 3 105 M21 s21 (Fig. 2).
FIG. 2. Rate constant between peroxynitrite and GAPDH. GAPDH (3.4 mM) was preincubated for 10 min with different concentrations of GSH (■) or cysteine (h) in 0.1 M pyrophosphate, 0.1 mM dtpa, pH 7.4, at 25°C and then exposed to 20 mM peroxynitrite. Aliquots were withdrawn, and GAPDH activity was assayed. Fi represents the fraction of protection of GAPDH in the presence of the scavenger ([S]); ks, the rate constant for the reaction of ONOO2 with the scavenger; and kd, the rate constant for the reaction of peroxynitrite with GAPDH. Plotting Fi[GAPDH]/(1 2 Fi)ks against [S] results in a straight line with the slope 1/kd, which allows the determination of kd (28, 30).
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Competition Experiments between GAPDH and Cytochrome c21 Cyt c21
GAPDH
Control 1ONOO2 1Competitor
Activity (mmol/min/mg)
% Initial activity
Concentration (mM)
% Initial concentration
120 22.0 45.2
100 18.3 37.7
80 58.6 73.8
100 73.2 92.3
Note. GAPDH (17 mM) and cytochrome c21 (80 mM) were exposed to 50 mM peroxynitrite in 0.1 M pyrophosphate, 0.1 mM dtpa, pH 7.4, for 10 min at 25°C in the absence or presence (1Competitor) of the other target molecule. Then, aliquots of the reaction mixtures were assayed by measuring the absorbance at 550 nm and GAPDH activity. The fraction of protection (Fi) for each target was determined and the rate constant for the GAPDH-peroxynitrite was determined independently in both assays as described previously (28, 29).
To further confirm the estimated rate constant between GAPDH and peroxynitrite obtained in Fig. 2, we performed a competition experiment between GAPDH and cytochrome c21 against peroxynitrite. Cytochrome c21 is oxidized to cytochrome c31 by peroxynitrite with a second-order rate constant of 1.32 3 104 M21 s21 at pH 7.4 and 25°C (34). The oxidation of cytochrome c21 by peroxynitrite was measured with and without GAPDH and, similarly, in the same experiment GAPDH inactivation by peroxynitrite was measured in the presence and absence of cytochrome c21 (Table II). From the data in Table II and using independent cal-
culations for either the fraction of protection of GAPDH or that of cytochrome c21, a rate constant of 2.0 3 105 M21 s21 was estimated, in good agreement with the data in Fig. 2. We evaluated whether the presence of substrate would protect the GAPDH active site from peroxynitrite-mediated inactivation. Glyceraldehyde 3-phosphate was able to protect GAPDH in a dose-dependent manner as shown in Fig. 3, reaching 100% protection at infinite substrate concentration (Fig. 3, inset). The other substrates, NAD1 and arsenate or phosphate, did not protect GAPDH (data not shown). It therefore seems probable that peroxyni-
FIG. 3. Peroxynitrite-mediated inactivation in the presence of substrate. GAPDH (0.52 mM) was incubated with different concentrations of glyceraldehyde 3-phosphate in 0.1 M pyrophosphate, 0.1 mM dtpa, pH 7.4, at 25°C for 5 min and then exposed to 30 mM peroxynitrite. (Inset) A Scachartd-like plot of the data.
GAPDH INACTIVATION BY PEROXYNITRITE
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FIG. 4. Influence of pH on peroxynitrite-mediated inactivation. GAPDH (1.4 mM) was incubated for 5 min in 0.1 M pyrophosphate, 0.1 mM dtpa at different pH levels and then exposed to 10 mM peroxynitrite. Aliquots were withdrawn, and GAPDH activity was assayed. Conditions were control (1), peroxynitrite (■), and 5 mM methionine (h).
trite and GAP interact with GAPDH at identical or adjacent sites on the enzyme. The inactivation of GAPDH by peroxynitrite was strongly pH-dependent (Fig. 4), with maximal inactivation obtained at alkaline pH. However, a significant fraction of the enzyme (;15%) was still inactivated at acidic pH. Methionine was able to protect GAPDH from inactivation mostly at acidic pH, in agreement with its reaction with peroxynitrous acid (35). During peroxynitrite-mediated GAPDH inactivation there was enzyme thiol oxidation (Fig. 5). While each monomer contains up to four thiols (1) (Fig. 5), only up to two thiols per enzyme monomer disappeared in the peroxynitrite concentration range of 0 – 40 mM, with only one critical thiol responsible for .90% enzyme inactivation as shown by the Tsou plot (36), where remaining enzyme activity was plotted against sulfhydryl loss (Fig. 5, inset). Arsenite and dithiothreitolmediated reduction of oxidized thiols permitted determination of the reversibility of sulfhydryl oxidation and aided identification of thiol oxidation states after peroxynitrite treatment (12, 37). Arsenite reduces sulfenic acid to sulfhydryl but does not reduce disulfides (37), while DTT reduces disulfides to thiols. Peroxynitrite-mediated oxidation of GAPDH thiols resulted in a product resistant to arsenite and only 15% DTT-recoverable (Table III). In contrast, iodosobenzo-
ate-mediated GAPDH thiol oxidation was 71% reduced by arsenite, suggesting a significant formation of sulfenic acid with this reagent as was previously shown by Parker and Allison (38). For comparative purposes with peroxynitrite reactivity, exposure of GAPDH to authentic •NO anaerobically (Fig. 6) resulted in a slow decrease in enzyme activity. Fitting this data to an exponential function, a second-order rate constant between •NO and GAPDH of 11.2 6 0.5 M21 s21 at pH 7.2 at 25°C is estimated. Preincubation of GAPDH with DTT completely protected against •NO-mediated inactivation. Postincubation with either DTT or arsenite resulted in some recovery of enzymic activity (Table III). On the other hand, under aerobic conditions where authentic •NO evolves to dinitrogen trioxide (N2O3), GAPDH was rapidly inactivated (in less than 30 s) to 60% of initial activity (Table III), with preincubation with GAP or DTT being completely protective. The addition of DTT to aerobic •NO-inactivated GAPDH caused a total reactivation of the enzyme (Table III). DISCUSSION
In this study we demonstrate that GAPDH is highly sensitive to inactivation by peroxynitrite. The high sensitivity is due to the high second-order rate con-
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FIG. 5. Sulfhydryl oxidation during GAPDH inactivation by peroxynitrite. GAPDH (2 mM) was incubated for 5 min in 0.1 M pyrophosphate, 0.1 mM dtpa, pH 7.4, at 25°C with different concentrations of peroxynitrite. Then aliquots were withdrawn, and GAPDH activity was assayed (■). Sulfhydryls were quantitated using the DTNB reaction in the presence of 1% SDS (h). (Inset) Fractional activity against oxidized sulfydryls per enzyme monomer.
stant of (2.5 6 0.5) 3 105 M21 s21 obtained for the GAPDH–peroxynitrite reaction and the 4:1 (peroxynitrite:GAPDH tetramer ratio) inactivation stoichiometry. The sensitivity of this enzyme compares well with that of others having oxidant-sensitive active sites, including alcohol dehydrogenase (31), aconitase (28), and glutathione peroxidase (39). In line with this concept, it has been recently shown that in Escherichia coli, the family of [4Fe-4S] cluster-containing dehydratases (aconitase, 6-phosphogluconate dehydratase, and fumarase A) and GAPDH were primary targets of peroxynitrite-mediated inactivation of enzymes (15). Peroxynitrite-mediated inactivation of GAPDH involving interactions at the active site can be concluded by the total protection afforded by excess substrate (Fig. 3). Substrate binding involves thioester bond formation between GAP and the essential SH groups of the enzyme, thus precluding peroxynitrite attack on the thiol. Indeed, inactivation of GAPDH by peroxynitrite consists of the oxidation of sulfhydryl groups of the enzyme (Fig. 5), one of which is responsible of most of the enzyme activity loss.
It is worth noting that the reaction of peroxynitrite with each subunit of GAPDH, which should primarily rely on interactions with a single critical thiol (Fig. 5), is one order of magnitude faster (6 3 104 M21 s21 per monomer) than that with cysteine, glutathione, and the single thiol group of bovine serum albumin (BSA) (12, 20, 22). This high rate constant between peroxynitrite and GAPDH may be dependent on the strongly hydrophilic and positively charged environment of the enzyme active site which would favor attraction of peroxynitrite toward the mercaptide (1, 40). Interestingly, one critical thiol group of GAPDH (Cys149) has been also shown to react with H2O2 with rates more than a hundred times faster than with the single thiol group of BSA at pH 7.0 (.100 M21 s21 (40) versus ;1 M21 s21 (12), respectively), confirming the unique reactivity of this residue. The preferential direct reaction of peroxynitrite with the thiol group of the enzyme is further supported by the protection afforded by cysteine, DTT, and methionine (and not by hydroxyl radical scavengers) and the pH profile of inactivation (12) (Table I and Fig. 4).
GAPDH INACTIVATION BY PEROXYNITRITE TABLE III
Reductive Recovery of Peroxynitrite, Nitric Oxide, and Iodosobenzoate-Oxidized GAPDH Condition
Reductant
% Initial activity
% Recovery
1ONOO2 1ONOO2 1ONOO2 1•NO (anaerobic) 1•NO (anaerobic) 1•NO (anaerobic) 1•NO (aerobic) 1•NO (aerobic) 1Iodosobenzoate 1Iodosobenzoate
— 1Arsenite 1DTT — 1Arsenite 1DTT — 1DTT — 1Arsenite
3.1 6 0.2 3.5 6 0.3 18 6 1 12 6 1 36 6 1 43 6 2 60 6 2 105 6 3 6.5 6 0.3 73 6 1
— 0.4 15 — 27 35 — 100 — 71
Note. GAPDH (2.7 mM) was exposed to 40 mM peroxynitrite, 200 mM •NO or 27 mM iodosobenzoate in 0.1 M pyrophosphate, 0.1 mM dtpa, pH 7.4, for 10 min (peroxynitrite and iodosobenzoate) or 30 min (•NO) at 25°C. Then, incubations with sodium arsenite or DTT were performed as indicated under Materials and Methods. Samples were immediately assayed for GAPDH activity. The percentage of recovery was calculated.
Although maximal inactivation was obtained at alkaline pH, some GAPDH inactivation was seen at acidic pH (Fig. 4), which is most likely dependent on the formation of secondary products from peroxynitrous acid (11, 20), which can oxidize sulfhydryls via a oneelectron oxidation mechanism (22). This reaction predominantly occurs with the thiolate form of the sulfhydryl, which in the case of GAPDH may be relevant even at acidic pH (i.e., 6.0) due to the low pKa of the critical thiol group of about 7.5 (40). This secondary mechanism can also explain recent results on the detection of thiyl radical in apo-GAPDH treated with 1,3-morpholinosydnonimine and peroxynitrite (16). The fast reaction between ONOO2 and CO2 (k 5 5.8 3 104 M21 s21 at 37°C; Ref. 33) leads to the formation of the nitrosoperoxocarbonate adduct, ONOOCO2 2, and this reaction would largely outcompete (;98%) the reaction of peroxynitrite with GAPDH under the conditions of Table I. However, protection from inactivation was marginal, which is consistent with the reported reactivity of ONOOCO2 2 toward thiols (33), causing enzyme inactivation in its own right. Although there is loss of two thiols per enzyme monomer, disulfide does not appear to be the main oxidation product as judged by the lack of reactivation by DTT (Table III). A minor amount of disulfide bond formation between Cys-149 and nearby Cys-153 may occur, although these residues can have only limited interactions as the side chain of Tyr-311 lies between the –SH groups of these two cysteines, inhibiting disulfide formation (41). Thus, considering that Cys-149 may be relatively isolated, it may have been oxidized by peroxynitrite to –SOH (sulfenic acid), –SO2H (sulfinic acid), or –SO3H (sulfonic acid) or to a combination of
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these. The mild oxidizing agents iodosobenzoate (Table III) or low concentrations of hydrogen peroxide (40) caused SH oxidation to sulfenic acid, which could be recovered by sodium arsenite. On the contrary, peroxynitrite oxidized the essential thiol of GAPDH toward higher oxidation states, sulfinic or sulfonic acid, because sodium arsenite was unable to recover activity from peroxynitrite-mediated GAPDH inactivation. This finding is similar to that with the single thiol group of BSA, where H2O2 oxidized it to an arsenitereducible product, while peroxynitrite oxidized it to a arsenite-resistant product (12). The mechanisms by which peroxynitrite promote formation of sulfinic and/or sulfonic acid derivatives in isolated protein thiols are not known at present. GAPDH has been implicated as one of the major intracellular targets of •NO, and the loss of GAPDH activity has been attributed to the linkage of NAD(H) to a cysteine residue of the active site. Herein, we have shown a slow inactivation process by authentic •NO on GAPDH under anaerobic conditions and in the absence of metals or NAD(H) (Fig. 6). This agrees with a slow nitric oxide-mediated oxidation of thiols under anaerobic conditions which may involve the formation of sulfenic acid derivatives (Table III), as previously reported (42). Aerobically, there was a more rapid •NOmediated inactivation, which would primarily depend on the formation of dinitrogen trioxide (N2O3), which
FIG. 6. Effect of •NO on GAPDH activity. GAPDH (1.8 mM) was incubated anaerobically with none (1), 200 mM •NO (Œ), and 200 mM • NO (h) in the presence of 1 mM DTT in 0.1 M Tris–HCl, 0.1 mM dtpa, pH 7.2, at 25°C, and activity was assayed in aliquots at different time points.
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promotes nitrosation of Cys-149 and loss of enzyme activity. Our study shows that GAPDH is highly reactive toward peroxynitrite, with this reactivity mostly dependent on the rather unique properties of the critical thiol group of the enzyme, Cys-149. GAPDH is an enzyme which is abundant in various tissues. For instance, in skeletal muscle, up to 10% of soluble protein is GAPDH and significant amounts also exist in heart muscle, brain, kidney, and liver (1). Thus, the ubiquity of GAPDH and its significant rate constant imply that GAPDH inactivation by peroxynitrite might be a relevant mechanism which may alter cellular glycolysis and/or gluconeogenesis as well as other functions recently ascribed to the enzyme (43). This process may occur even in the presence of biological levels of CO2, both because the reaction of cellular GAPDH-SH may compete well with the formation of ONOOCO2 2 , but also because ONOOCO2 is also able to efficiently oxi2 dize thiols. ACKNOWLEDGMENTS We thank Dr. Gerardo Ferrer for helpful comments and critical reading of the manuscript. This work was supported by grants from CSIC, Uruguay, to J.M.S. and CONICYT, Uruguay, and SAREC, Sweden, to R.R.
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