Department of Pharmacology and Pharmacotherapy, Toxicology Section, University of Pécs, Medical School, Pécs, Hungary
1 To whom correspondence should be addressed at Department of Pharmacology and Pharmacotherapy, Toxicology Section, University of Pécs, Medical School, Szigeti út 12, H-7624 Pécs, Hungary. Fax: 3672536218. E-mail: zoltan.gregus{at}aok.pte.hu.
Received February 11, 2005; accepted March 18, 2005
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ABSTRACT |
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Key Words: arsenate; glyceraldehyde-3-phosphate dehydrogenase; koningic acid; glutathione; NAD; reduction.
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INTRODUCTION |
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It has recently been shown that human red blood cells (RBC) possess a PNP-independent AsV reductase activity (Németi and Gregus, 2004), and circumstantial evidence has been presented that this activity depends on the availabilities of glutathione (GSH) and NAD or NADP. Furthermore, it has been proposed that this activity is linked to one or more enzymes in the glycolytic pathway between glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and enolase. The previous work (Németi and Gregus, 2005
) has further characterized this AsV reductase activity in human RBC lysate and rat liver cytosol, presented direct evidence for its NAD- and GSH-dependent nature, and confirmed the suggestion that it is associated with glycolysis. This work demonstrated that in addition to NAD, the glycolytic substrates fructose-1,6-bisphosphate (Fruc-1,6-BP), 3-phosphoglycerate (3-PGA), and 2- phosphoglycerate (2-PGA) supported reduction of AsV the greatest. The strong dependence of AsV reduction on NAD makes GAPDH a candidate enzyme with AsV reductase activity, because GAPDH is the only glycolytic enzyme, for which NAD is both a substrate and an activator (Tilton et al., 1991
). However, the marked stimulation of the hemolysate- and cytosol-mediated AsV reduction by 3-PGA and 2-PGA (which can readily be converted to 3-PGA) makes phosphoglycerate kinase (PGK) also a candidate, because 3-PGA is a substrate for PGK, and because in the glycolytic pathway PGK is linked to GAPDH through their common substrate 1,3-bisphosphoglycerate (1,3-BPG) (see scheme in Németi and Gregus, 2005
). Moreover, GAPDH can use AsV instead of inorganic phosphate (Pi) when converting glyceraldehyde-3-phosphate (Ga-3-P) into 1-arseno-3-phosphoglycerate, instead of 1,3-BPG.
The aim of the present study was to determine if GAPDH, PGK, or both enzymes can function as AsV reductase. For this purpose, we first tested whether the two purified enzymes supplemented with various combinations of their nucleotide and glycolytic substrates could carry out the reduction of AsV in the presence of GSH. These experiments have clarified that it is GAPDH and not PGK that catalyzes the reduction of AsV in the presence of GSH, NAD, and its glycolytic substrates. Therefore, we performed further studies in order to characterize the AsV reductase activity of this enzyme as well as to test, using the GAPDH inhibitor koningic acid (KA; Beisswenger et al., 2003; Kato et al., 1992
; Sakai et al., 1988
), whether GAPDH contributes to reduction of AsV in human RBC lysate, rat liver cytosol, and intact human erythrocytes.
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MATERIALS AND METHODS |
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Assays of AsV reduction and GAPDH activitygeneral conditions.
If otherwise not specified, assays were carried out and substances were dissolved in sucrose buffer, containing 250 mM sucrose, 25 mM HEPES, 5 mM MgCl2, 2 mM EGTA, pH 7.4. AsV reductase assays were run in 300 µl final volume, in microcentrifuge tubes kept in a shaking water bath at 37°C. They were started by addition of AsV (50 µM) and terminated by sequential addition of 100 µl 25 mM CdSO4 solution followed by 100 µl 1.5 M perchloric acid solution containing 25 mM HgCl2. The rationale for this procedure is given in the adjoining paper (Németi and Gregus, 2005). The incubates thus treated were stored at 80°C until arsenic analysis. AsV reductase activity was expressed as the amount of AsIII formed per minute and quantity of enzyme source (e.g., unit GAPDH, ml packed RBC, mg cytosolic protein).
The GAPDH activity was assayed spectrophotometrically based on the decrease of NADH concentration (0.25 mM) during the GAPDH-limited conversion of 3-PGA (5 mM) to Ga-3-P in the presence of excess PGK (1 U) and ATP (5 mM). This procedure measures the rate of the reverse reaction rather than the forward reaction, which occurs during glycolysis. The assay was carried out in 1 ml final volume, in the spectrophotometer cell, at 25°C, and was started by addition of GAPDH or its source (e.g., diluted hemolysate, cytosol). The enzyme activity was calculated from the change of absorbance (A/min at 340 nm), taking 6220 M1 as the molar extinction coefficient of NADH340nm. The GAPDH activity was expressed as unit per volume (U/ml, corresponding to µmol NADH utilized per min and ml) or U per quantity of enzyme source (e.g., ml packed RBC, mg cytosolic protein). For both assays, more details are given below.
Purified enzyme experiments.
Stock solutions from the commercially obtained GAPDH and PGK were prepared freshly and kept in ice until use within 2 h. The GAPDH activity in the stock solution was regularly quantified spectrophotometrically as described above.
When analyzing the role of GAPDH and PGK as well as their substrate supply in AsV reduction, the incubations contained these enzymes (both at 2 U/ml concentration): Ga-3-P (1 mM), NAD (1 mM), and ADP (1 mM) (when testing the forward reaction), or 3-PGA (1 mM), NAD (1 mM), and ATP (1 mM) (when testing the reverse reaction). The incubation was started by adding GSH (6 mM), GAPDH, PGK, and AsV (50 µM), and continued for 10 min. When characterizing the AsV reducing activity of purified GAPDH, incubations were performed in the presence of Ga-3-P and NAD (both 1 mM). The incubation was started by adding GSH (6 mM) and GAPDH (2 U/ml) followed by AsV (50 µM), and it lasted for 10 min. To determine the effect of koningic acid (dissolved in dimethyl sulfoxide (DMSO) on the AsV-reducing activity of purified GAPDH, the enzyme (2 U/ml) was preincubated for 10 min with koningic acid (2.550 µM) or DMSO (not exceeding 1% final concentration). Thereafter, GSH (6 mM), Ga-3-P (0.5 mM), NAD (1 mM) were added, followed by AsV (50 µM), and the incubation was continued for 10 min. To measure the effect of koningic acid on the classical enzymatic activity of GAPDH, the enzyme (2 U/ml) was incubated with koningic acid (or DMSO) for 10 min, and then its activity was measured immediately, using the spectrophotometric assay described above.
RBC experiments.
This research was approved by the Regional Scientific Research Ethics Committee of the University of Pécs, Center for Medical and Health Sciences. Blood (approximately 5 ml) was collected from healthy human volunteers after informed consent. For the experiments with hemolysate, the washed RBC were prepared with and incubated in sucrose buffer as described in the previous work (Németi and Gregus, 2005). For the experiments with intact RBC, however, the RBC suspension was prepared and incubated in a chloride-free gluconate buffer, containing 150 mM sodium gluconate, 10 mM HEPES, 5 mM glucose, pH 7.4. This was necessary, because chloride inhibits the uptake of AsV by the erythrocytes (Németi and Gregus, 2004
).
To determine the effect of koningic acid on the PNP-independent AsV reductase activity of hemolysate, erythrocytes (50 µl packed cells) were preincubated at 37°C for 10 min in sucrose buffer with a PNP inhibitor (BCX-1777, 20 µM), detergent (Nonidet P-40, 0.067%), glucose oxidase (2 U), and koningic acid (2.5100 µM) or DMSO. Thereafter, the incubation was started by adding GSH (6 mM), buffer or Fruc-1,6-BP (1 mM) plus NAD (1 mM) or 3-PGA (1 mM) plus NAD, followed by AsV (50 µM), and was continued for 2.5 min. To measure the effect of koningic acid on the GAPDH activity of hemolysate, RBC (50 µl packed cells) were incubated with koningic acid (or DMSO) for 10 min at 37°C in a final volume of 0.3 ml of sucrose buffer in the presence of detergent (Nonidet P-40, 0.067%). Thereafter, the incubate was diluted with ice-cold buffer approximately 17-fold, and its GAPDH activity was immediately assayed.
To determine the effects of koningic acid on AsV reductase activity of intact RBC, erythrocytes (15 µl packed cells) were preincubated with koningic acid (150 µM) or DMSO for 20 min in gluconate buffer. Thereafter, the incubation was started by adding buffer, pyruvate (0.25 mM), or ferricyanide (0.25 mM) followed by AsV (50 µM), and was continued for 30 min. The incubations were stopped as described above except that the CdSO4 solution contained 1% Triton X-100. To measure the effect of koningic acid on the GAPDH activity of intact erythrocytes, RBC were incubated in gluconate buffer with koningic acid for 20 or 50 min (corresponding to time points when incubations with AsV would start and end). Therefater, RBC were pelleted by brief centrifugation (10,000 x g, 30 sec), and the supernatant was carefully removed. The cells were resuspended in 33 volumes of ice-cold saline and centrifuged again. After careful supernatant removal, RBC were lysed by adding 99 volumes of water and brief sonication. The GAPDH activity of RBC lysate thus prepared was immediately assayed spectrophotometrically.
Cytosol experiments.
To prepare rat liver cytosol, male Wistar rats weighing 250270 g and maintained under standardized conditions were used. All procedures were carried out according to the Hungarian Animals Act (Scientific Procedures, 1998), and the study was in agreement with the rules of the Ethics Committee on Animal Research of the University of Pécs. The cytosolic fraction was prepared by differential centrifugation of the liver homogenate made in sucrose buffer as described in the preceding paper (Németi and Gregus, 2005). Cytosolic protein concentration was quantified by the bicinchoninic acid method according to Brown et al. (1989)
.
To determine the effects of koningic acid on the PNP-independent AsV reductase activity of cytosol, rat liver cytosol (5 mg protein/ml) was preincubated for 10 min in sucrose buffer with a PNP inhibitor (BCX-1777, 20 µM), glucose oxidase (2 U), and koningic acid (2.5100 µM) or DMSO. Thereafter, the incubation was started by adding GSH (10 mM), buffer or Fruc-1,6-BP (1 mM) plus NAD (1 mM), or 3-PGA (1 mM) plus NAD, followed by AsV (50 µM), and was continued for 2.5 min. To measure the effects of koningic acid on the GAPDH activity of cytosol, rat liver cytosol (5 mg protein/ml) was incubated with koningic acid (2.5100 µM) or DMSO for 10 min. Thereafter, the GAPDH activity of the cytosol was immediately assayed, using the spectrophotometric procedure.
Arsenic analysis.
The incubates having been subjected to protein precipitation were centrifuged at 10,000 x g, 4°C for 10 min. AsIII and AsV in the resultant supernatants were separated and quantified by HPLC-hydride generation-atomic fluorescence spectrometry (HPLC-HG-AFS), the details of which have been given elsewhere (Gregus et al., 2000; Németi et al., 2003
).
Statistics.
Data were analyzed using one-way ANOVA followed by Duncan's test or Students' t-test with p < 0.05, as the level of significance.
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RESULTS |
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Figure 1 demonstrates that, when 3-PGA, ATP, and NAD were the added substrates, the mixture of GAPDH (rabbit) and PGK in the presence of GSH reduced AsV at a rate of 31.5 ± 5.0 pmol/minute. Omitting any one of the components from the complete incubations practically abolished AsIII formation, although the lack of exogenous NAD resulted in strong but incomplete inhibition of AsV reduction. Replacement of NAD with NADH or ATP with ADP in the complete incubation mixture also markedly diminished AsV-reducing activity.
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Reduction of AsV to AsIII by Purified GAPDH
Since the observations presented above strongly suggested that AsV reduction was carried out by GAPDH, we tested if this enzyme alone supported by its two substrates (i.e., Ga-3-P and NAD) was capable of reducing AsV in the presence of GSH. As demonstrated in Figure 3, the reduction rate increased proportionately to the concentration of rabbit muscle GAPDH up to 2 units/ml. Above this concentration, the reduction rate increased less than proportionately. The human GAPDH seemed to have somewhat higher AsV-reducing activity than the rabbit muscle enzyme.
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In the absence of KA, the GAPDH activity of the hemolysate was 19.6 ± 1.86 U/ml packed cell. Preincubation with KA diminished GAPDH activity of the RBC lysate in a concentration-dependent fashion (Fig. 8, top), causing an approximately 20% inhibition at a concentration as low as 2.5 µM, and near complete inhibition at 50100 µM. AsIII formation rates in the hemolysate without substrates, with Fruc-1,6-BP plus NAD, and with 3-PGA plus NAD were 0.46 ± 0.09, 6.15 ± 0.87, and 12.3 ± 0.71 nmol/min/ml packed cell, respectively. Without added substrate, preincubation with KA decreased the AsV-reducing activity significantly at 10 µM concentration and above (Fig. 8, bottom); however, 1720% activity remained in the presence of even 50100 µM KA. The Fruc-1,6-BP plus NAD and 3-PGA plus NAD-stimulated AsV reduction exhibited much higher sensitivity toward KA. The diminution in AsIII formation caused by preincubation with KA was significant at 10 µM concentration and above; AsV reduction was barely detectable at 25 µM KA and was abolished by KA at 50 µM and above.
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DISCUSSION |
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The mix of GAPDH and PGK formed AsIII from AsV when supplemented with Ga-3-P, NAD, and ADP (Fig. 2). Under this condition, GAPDH converts Ga-3-P to 1,3-BPG (in the presence of Pi) or 1-arseno-3-phosphoglycerate (in the presence of AsV), which in turn may be converted by PGK to 3-PGA (see insert in Fig. 2). Two findings indicate that PGK in this system does not contribute to AsV reduction. First, omission of PGK alone did not influence the AsV reductase activity (Fig. 2). Second, omission of ADP alone, which would prevent PGK from converting the GAPDH-produced 1,3-BPG (or 1-arseno-3-phosphoglycerate) to 3-PGA, even enhanced the reduction of AsV (Fig. 2). However, omission of PGK did abolish the AsV reductase activity of the mix of GAPDH and PGK, when this enzyme pair was supplemented with 3-PGA, ATP, and NAD (Fig. 1). Under this condition, PGK converts 3-PGA to 1,3-BPG for GAPDH, which in turn can 1-dephosphorylate 1,3-BPG and bind the resultant 3-phosphoglyceroyl group, but cannot reduce it to Ga-3-P in the absence of NADH. In the light of the findings discussed so far, the role of PGK under the conditions shown in Figure 1 can be interpreted as that of an auxiliary enzyme merely providing substrate for GAPDH, the actual NAD- and GSH-dependent AsV reductase. This interpretation is confirmed by the observation that GAPDH alone is capable of catalyzing reduction of AsV to AsIII in the presence of three indispensable ingredients, namely its glycolytic substrate Ga-3-P (Fig. 4, left), NAD (Fig. 4, right), and GSH (Fig. 5).
AsV reductase activity of purified GAPDH exhibited distinct concentration dependence on GSH, NAD, and Ga-3-P. The rate of the GAPDH-catalyzed AsV reduction kept increasing with an increase in GSH concentration (Fig. 5) similarly to that seen with the rate of AsV reduction catalyzed by hemolysate or cytosol (Fig. 1 in Németi and Gregus, 2005). The hyperbolic curve representing the concentration-dependent effect of NAD on GAPDH-catalyzed AsV reduction reflects most likely the saturation of the enzyme with this coenzyme at 1 mM, above which NAD causes no additional increase in AsV reduction (Fig. 4, right). To rationalize the peculiar concentration-dependent effect of Ga-3-P on the AsV reductase activity of GAPDH (Fig. 4, left), it should be considered that Ga-3-P is not only a substrate of GAPDH but also its allosteric inhibitor (Tomschy et al., 1993
). Furthermore, it should also be realized that out of necessity we used racemic Ga-3-P (i.e., a mixture of the enzymatically active D-isomer and the inactive L-isomer). It has been reported that at higher concentrations, the racemic Ga-3-P produced stronger inhibition of GAPDH activity than the pure D-isomer, indicating that the L-isomer is more potent allosteric inhibitor of the enzyme than the D-isomer (Tomschy et al., 1993
). Thus, the observed decline in the GAPDH-catalyzed AsIII formation from AsV above 0.5 mM Ga-3-P (Fig. 4, left) can most likely be ascribed to the inhibitory effect of Ga-3-P, especially of the L-isomer present in the commercially available racemic mixture. The inhibitory effect of Ga-3-P on AsV reduction by the hemolysate in the absence of exogenous NAD described in the previous paper (Németi and Gregus, 2005
) may, in part, originate from the presence of L-Ga-3-P also. In addition, when Ga-3-P is the added substrate, the enzyme produces NADH, which strongly inhibits the GAPDH-catalyzed AsV reduction even in the presence of 1 mM NAD (Fig. 6). Adenine nucleotides (i.e., AMP, ADP, and ATP) are known to weakly inhibit GAPDH by competing with NAD for binding (Nakamura et al., 1982
). This may account for the moderate inhibition of AsV reduction catalyzed by purified GAPDH (Fig. 6), hemolysate, or cytosol (Németi and Gregus, 2005
) brought about by these nucleotides. Furthermore, 2,3-bisphosphoglycerate (2,3-BPG) also inhibits GAPDH (Srivastava and Beutler, 1972
); this may explain why this compound decreased AsV reduction when added to hemolysate or rat liver cytosol (Németi and Gregus, 2005
).
The experiments carried out with KA also corroborate the role for GAPDH in the reduction of AsV to AsIII. The epoxide group-containing KA is a potent and specific inhibitor of GAPDH; it irreversibly inhibits the enzyme by forming a thioether bond with the active site cysteine at position 149, especially when the enzyme is complexed with NAD (Beisswenger et al., 2003; Kato et al., 1992
; Sakai et al., 1991
). As demonstrated in Figure 7, KA inhibited not only the classical enzymatic activity of GAPDH, but also its AsV reductase activity in a concentration-dependent manner. Nevertheless, Ga-3-P markedly decreased the inhibitory effect of KA on the GAPDH-catalyzed AsV reduction, when the enzyme was preincubated in the combined presence of KA and Ga-3-P (Table 1). This protective effect of Ga-3-P against KA results from the fact that Ga-3-P competes with KA for binding to GAPDH and may account, at least in part, for the incomplete inhibition by KA of both the GAPDH and the AsV-reducing activities of hemolysate and cytosol (Figs. 8 and 9, respectively). Importantly, GSH did not protect GAPDH from inactivation by KA (Table 1), indicating that the thiol reactivity of KA is not universal. Furthermore, KA-induced inactivation of GAPDH depends not only on KA concentration, but also on the incubation time (Sakai et al., 1988
). Accordingly, the inhibition of GAPDH in intact RBC became more pronounced after longer incubations with KA (Fig. 10).
Demonstration that KA inhibits the AsV reductase activity of purified GAPDH permitted us to use KA as an experimental tool to assess the contribution of GAPDH to the PNP-independent reduction of AsV by the hemolysate, the liver cytosol, and the intact erythrocytes. The experiments with KA strongly suggest that the PNP-independent AsV reductase activity of the human lysed RBC and rat liver cytosol can almost solely be attributed to GAPDH under conditions that provide abundant NAD and glycolytic substrate supply for this enzyme (i.e., in the presence of NAD plus Fruc-1,6-BP or 3-PGA), because the GAPDH and AsV reductase activities decreased in parallel in both the hemolysate and the cytosol in response to increased concentration of KA, and when KA abolished the activity of GAPDH, formation of AsIII from AsV ceased (Figs. 8 and 9, respectively). However, when GAPDH was not provided with exogenous NAD and substrates, even 100 µM KA failed to inhibit AsV reduction by the hemolysate and the liver cytosol completely (Figs. 8 and 9, bottom panels), even though the GAPDH activities in these cell extracts were practically abolished. While the residual AsV-reducing activity in the presence of 100 µM KA was only 18% of control in the hemolysate (Fig. 8, bottom), in the cytosol it was as high as 60% (Fig. 9, bottom). Since both PNP and GAPDH activities in the rat liver cytosol were blocked (by BCX-1777 and KA, respectively), this finding supports the hypothesis put forward in the preceding paper (Németi and Gregus, 2005) that, besides GAPDH, there is another hitherto unidentified cytosolic enzyme, which is able to reduce AsV and is supported by GSH and NAD(P).
In intact human RBC, irrespective of whether the glycolysis was or was not stimulated with NADH oxidants (pyruvate or ferricyanide), KA was much more effective in inhibiting AsV reductase activity than in the hemolysate or the cytosol. For example, KA concentrations as low as 10 µM caused a near complete inhibition of AsIII formation in the intact erythrocytes (Fig. 10). As to the increased sensitivity of erythrocytes to KA, one may raise the possibility that these cells may concentrate the inhibitor, or GAPDH may be in a conformation in situ that is more susceptible to inhibition by KA. In addition, in the light of the discussion above, it may be speculated that GAPDH in intact erythrocytes is well supplied with NAD and glycolytic substrate, conditions which would give GAPDH a proportionally larger role in AsV reduction in these cells than in the hemolysate without optimal NAD and substrate supply. Nevertheless, it is to be emphasized that our observation on AsV reduction in RBC should not give the impression that erythrocytes play a significant role in AsV reduction in the body. Such a role is severely limited by Pi and chloride ions present in the plasma (but not in our incubation buffer), which inhibit erythrocytic uptake of AsV (Németi and Gregus, 2004).
The mechanism of the GAPDH-catalyzed AsV reduction is unknown. This work, however, provides some clues to put forward a hypothesis, which we present after briefly describing the catalytic cycle of this enzyme. GAPDH is composed of four identical subunits with one molecule of NAD bound strongly to each. During glycolysis, this enzyme catalyzes both the oxidation of Ga-3-P and the incorporation of Pi into Ga-3-P to produce 1,3-BPG. This process involves four main steps. In step 1, Ga-3-P covalently binds to Cys149 of the enzyme forming a thiohemiacetal (Harris and Waters, 1976; Nagradova, 2001
; Nagradova and Schmalhausen, 1998
). In step 2, the bound Ga-3-P is oxidized into 3-phosphoglyceric acid still bound to Cys149, now with a thioester bond, while NAD is reduced to NADH. In step 3, the NADH formed in step 2 is replaced by NAD. This is necessary because NAD binding induces a conformational change, which will dislocate the phosphate group of the 3-phosphoglyceroyl moiety from the binding site for Pi making it accessible for Pi (Nagradova, 2001
) or, in our case, for AsV. In step 4, the thioester bond is cleaved by Pi (termed phosphorolytic cleavage), releasing the 3-phosphoglyceroyl moiety as 1,3-BPG from Cys149 of GAPDH. Instead of Pi, AsV can also cleave this bond (by arsenolytic cleavage) to produce the purportedly unstable 1-arseno-3-phopshoglycerate, instead of 1,3-BPG.
Our observations presented here are compatible with the following tentative suggestions: (1) the enzyme is ready to catalyze the reduction of AsV when it forms the 3-phosphoglyceroyl-enzyme : NAD complex (i.e., it carries bound NAD and the 3-phosphoglyceroyl group bound via a thioester bond to Cys149), and (2) the GAPDH-mediated AsV reduction takes place during, or as a consequence of, the arsenolytic cleavage of the thioester bond formed between the enzyme's Cys149 and the 3-phosphoglyceroyl group of the substrate. The first suggestion is supported by the observation that GAPDH catalyzed the reduction of AsV in the presence of PGK, ATP, 3-PGA, and NAD (Fig. 1). Under this condition, PGK uses ATP to phosphorylate 3-PGA into 1,3-BPG (see insert in Fig. 1), which in turn will acylate GAPDH at Cys149 while releasing Pi. Thus, the critical 3-phosphoglyceroyl-enzyme : NAD complex is formed, which cannot undergo reduction to Ga-3-P because NADH is absent, but can undergo phosphorolysis or arsenolysis. As to the second suggestion, a prerequisite for the phosphorolysis (or arsenolysis) is that the NADH formed as a result of substrate oxidation (step 2) be replaced by NAD. The arsenolytic (or phosphorolytic) cleavage is strongly inhibited by NADH (Nagradova and Schmalhausen, 1998), and so is the GAPDH-catalyzed AsV reduction (Fig. 6), supporting the importance of the arsenolytic cleavage in the reduction of AsV.
When proposing a mechanism for AsV reduction by GAPDH, it is important to note that three thiols cooperate in the process catalyzed by microbial AsV reductases. The E. coli (Martin et al., 2001) and yeast (Mukhopadhyay et al., 2000
) AsV reductases are monothiol enzymes, for which GSH and glutaredoxin provide the two additional thiol groups, whereas in the Staphylococcus aureus enzyme all three thiols belong to the enzyme (Messens et al., 2002
). In this latter enzyme, two thiols bind and reduce AsV to AsIII, while they form a disulfide bond, which will be reduced by the third thiol. The disulfide bond then formed is reduced by thioredoxin, completing a process termed thiol-disulfide cascade. In the microbial reductases, the catalytically important thiol-group is activated by a nearby positive charge. The S. aureus enzyme may functionally model GAPDH, because three thiols are also present and located in GAPDH near the site of the phosphorolytic/arsenolytic cleavage. One of these is GSH, which is associated with GAPDH very strongly (Krimsky and Racker, 1952
) and is claimed to protect the 3-phosphoglyceroyl-enzyme : NAD complex against cleavage of the thioester bond by water (i.e., hydrolysis) (Kuzminskaya et al., 1993
). Another thiol is the enzyme's Cys153, which is located at the active site of the GAPDH close to the catalytically important Cys149 in the same
-helix, and the third is Cys149, which becomes free upon the arsenolytic cleavage of the thioester bond. Like the microbial enzymes, the active site of GAPDH also contains a thiol-activating positive charge on the imidazole ring of His176 (Nagradova and Schmalhausen, 1998
). Thus, the three thiols might contribute to the reduction of AsV when it is in 1-arseno-3-phosphoglycerate, or after it is released from the latter unstable compound. The formed AsIII may then be released and complexed by GSH present in large excess in the ambient solution. GSH may also serve to reduce the disulfide bonds formed during AsV reduction. Theoretically, it is also possible that the contribution of GAPDH to the reduction of AsV is indirect, and GSH directly reduces AsV while being in mixed anhydride with 3-PGA as 1-arseno-3-phosphoglycerate. Even then, GAPDH is required, because formation of 1-arseno-3-phosphoglycerate must precede the reduction of AsV. Whether the role of GAPDH in AsV reduction is direct or indirect, the inhibitory effect of KA on the GAPDH-mediated AsV reduction is compatible with our hypotheses, as covalent binding of KA to Cys149 (Sakai et al., 1991
) prevents formation of the 3-phosphoglyceroyl-enzyme and 1-arseno-3-phosphoglycerate. Nevertheless, more research is needed on the mechanism of GAPDH-mediated AsV reduction.
In summary, this work demonstrates that GAPDH can fortuitously function as an AsV reductase, provided NAD, glycolytic substrate, and GSH are available. We hypothesize that GAPDH-catalyzed AsV reduction takes place during, or as a result of, the arsenolytic cleavage of its substrate from the thioester bond between the active site cysteine and the substrate. As studies with the specific GAPDH inhibitor KA indicate, GAPDH is exclusively responsible for the PNP-independent AsV reductase activity in human erythrocytes and significantly contributes to the AsV reduction in rat liver cytosol. Further research should clarify whether or not GAPDH contributes to the reduction of AsV to AsIII in vivo.
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NOTES |
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ACKNOWLEDGMENTS |
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