Activity of TR Towards Se-Containing Substrates
The importance of the Sec residue to catalysis has been shown in several studies and mutation of Sec to Cys causes a large drop in the rate of Trx reduction, 175- to 550-fold in kcat
). In contrast, when we tested the Sec
Cys mutant of TR using selenocystine as the substrate, the activity was only 3.7-fold lower than that of the wild type (WT) enzyme as shown by the data in . Similarly, there is only a ~3-fold difference in activities between the mammalian enzyme and DmTR, a TR that has a Cys residue in the 2nd
position of the dyad instead of Sec. The data in also shows that the truncated enzyme missing the C-terminal reaction center reduces the diselenide bond of selenocystine at a dramatically lower rate, while the mutant in which only Cys1
is present in the C-terminal reaction center reduces selenocystine at an intermediate rate. This demonstrates that the enzyme uses a pathway for reduction of this substrate that depends on the use of the C-terminal reaction center, but this mechanism must be distinct from the pathway that the enzyme uses for the reduction of the disulfide bond of Trx. This distinctiveness is demonstrated by: (i) the fact that substitution of Sec2
results in a large decrease in the rate of reduction of Trx, (ii) the truncated enzyme in which only Cys1
is present will not reduce Trx at all (data not shown). These two points are in marked contrast when selenocystine is the substrate as shown in .
Activities of Various TRs Towards Selenocystine and Cystinea
We also tested sodium selenite as a substrate for the WT enzyme and the same mutants discussed above and the results are summarized in . A similar, but not identical pattern is exhibited. Similar to when selenocystine is used as the substrate, the Cys2-mutant enzyme has only ~ 2-fold less activity than the WT enzyme when selenite is the substrate. However the truncated enzyme missing the C-terminal reaction center (mTRΔ8) still had very significant activity with selenite – only 6-fold lower activity than the WT enzyme, and the mutant missing Sec2, but still containing Cys1 has the same activity as the truncation mutant where both Cys1 and Sec2 are absent. These last two results are different than when selenocystine is the substrate.
Activities of Various TRs Towards Selenitea
The data in and also show that DmTR reduces selenocystine and selenite at considerable rates. This observation together with the fact that the Cys2
-mutant of the mammalian enzyme reduces these Se-containing substrates at rates comparable to the WT enzyme demonstrates that the nucleophilicity of Sec, relative to Cys, is not an important factor for some reactions catalyzed by TR, as we have previously contended (26
). Further, the presence of Sec in TR is not entirely responsible for the broad substrate specificity of the enzyme.
The data presented above leads to the interesting observation that Se can be removed from the enzyme, resulting in only a small decline in substrate turnover rate, if a Se atom is present in the substrate.
This model can be rationalized by understanding the types of bonds that the N-terminal reaction center can reduce. As shown in , the C-terminal reaction center reduces macromolecular Trx and becomes oxidized, forming a cyclic S1
bond as an 8- membered ring. This selenosulfide bond is essentially an internal substrate for the N-terminal redox center and can be reduced by the N-terminal reaction center because it is polarized and has a low pKa
leaving group (Se) (36
). As has been previously demonstrated, the disulfide bond of DTNB can be reduced directly by the N-terminal reaction center. This is because the S-S bond of DTNB is highly polarized due to the presence of symmetrical 2-nitrobenzoate groups bonded to each S atom, and as a result of this polarization this S-S bond is highly electrophilic. In addition, this strong polarization results in a leaving group pKa
of the thionitrobenzoate anion of 4.75 (37
). These two observations lead us to hypothesize that the N-terminal reaction center prefers to reduce substrates of the form S-Y, where Y is a good leaving group. We explain this hypothesis in mechanistic terms in showing our proposed mechanism for the reduction of selenocystine by TR. This mechanism explains why Se-containing substrates are turned over by the Cys2
-mutant at only slightly lower rates than the Sec2
-WT enzyme, in direct contrast when Trx is reduced by the Cys2
-mutant. In the case in which Sec2
is mutated to Cys2
with Trx as the substrate, the mutant enzyme still utilizes a ring formation pathway but now an 8-membered S1
ring is formed as an intermediate. This type of disulfide is unreactive towards thiol-disulfide exchange with the N-terminal reaction center, as we have already previously demonstrated (26
). Whereas when selenocystine is the substrate (), the enzyme could utilize a pathway that bypasses ring formation, but still allows for formation of a S1
bond (of the type S-Y, though in this case the selenosulfide bond that forms is between enzyme and substrate instead of an internal selenosulfide bond), and this S1
bond is reactive towards thiol/disulfide exchange with the N-terminal reaction center.
Figure 3 Comparison of the current, accepted mechanism of the reduction of Trx (A) with our proposed alternate mechanistic pathways with Se-containing substrates (B and C). In (A) The Se atom of Sec2 attacks the disulfide bond of Trx to form a mixed selenosulfide (more ...)
The mechanistic situation with selenite as substrate is somewhat different. Selenite, like the S-S bond of DTNB, is highly electrophilic and the data in shows that it can be reduced by the N-terminal reaction center directly, with only a small loss in activity compared to the full-length enzyme when Sec2 is present. The electrophilicity of selenite is a direct consequence of the Se atom, as we were unable to detect activity using sulfite as the substrate (data not shown). Because the truncated enzyme where only Cys1 is present had the same activity as the Δ8 enzyme, the mechanism shown in is unlikely to be used for selenite. Instead, the thiolate of CysIC could attack the mixed diselenide bond between enzyme and substrate directly. The proposed mechanism also bypasses ring formation (), but in a different way than for selenocystine. The reason for higher activity for the enzymes with an intact C-terminal reaction center is that this allows for further polarization of the bond between enzyme and substrate, whether the 2nd residue of the dyad is Cys or Sec. We explain the difference in enzyme mechanisms for selenocystine and selenite as being due to the difference in polarization in the bond formed between enzyme and substrate (compare the middle panels of ). The three oxygen atoms on selenite confer significant polarization to the mixed diselenide bond, allowing for direct attack by CysIC, whereas in the case of selenocystine, a relatively unpolarized diselenide would form between enzyme and substrate and this diselenide bond is unreactive towards exchange with the N-terminal reaction center. Thus the S atom of Cys1 is needed to form the required S-Y bond. This idea is supported by the fact that the Δ8 enzyme cannot directly reduce selenocystine.
Further evidence that the mechanisms of these two substrates are different is evidenced by the pH vs. activity profiles. The profile for selenite is broad with a pH optimum between pH 6 and 7, while the profile for selenocystine is also broad, but the optimum is between 7.5 and 8. These profiles are shown in the Supporting Information
as Figures S1A and S1B
, respectively. It is also interesting to view the profiles of these two substrates for the mammalian Δ8 enzyme so that the effect of pH on activity can be determined for the N-terminal reaction center only. For selenite, the pH optimum is near pH 6.0 and the profile is significantly sharper than the full-length enzyme. For selenocystine, the pH optimum is near 8.0 for the truncated enzyme (shown in the Supporting Information
as Figures S2A and S2B
, respectively). As reported previously, the pH optimum of the Δ8 enzyme with DTNB as substrate is also shifted significantly towards acidic pH (26
). Both selenite and DTNB are highly electrophilic compounds. It is tempting to infer that the reason for the occurrence of Se in the mammalian enzyme is that there is a certain electrophilic threshold required by the N-terminal reaction center for substrate reduction (note sulfite is a poor substrate), whether it is Se in an external substrate like selenite, or Se in the S1
bond of the C-terminal selenosulfide motif (internal substrate). The electrophilicity of Se is a well established principle in organic chemistry (38
). This idea is complementary to the leaving group concept for Se in TR we introduced previously.
Our proposed leaving group concept is further supported by the result in showing that selenocystine is a much better substrate in comparison to cystine. These two substrates are highly similar in terms of dihedral angles, overall charge, and size (39
). A major difference between these two small molecule substrates is leaving group pKa
upon Se–Se or S–S bond scission, as the pKa
of a selenolate is ~5.2 versus ~8.3 for that of a thiolate (36
). Thus cystine unlike DTNB, could not be reduced by the truncated Δ8 enzyme (direct reduction by the N-terminal reaction center), because its S-S bond is not polarized and lacks a good leaving group. Our proposed mechanisms also explain why cystine cannot be reduced by the WT-enzyme. If the pathway outlined in is used to reduce cystine, a mixed S1
bond would form between enzyme and substrate and this is not of the type S-Y. This S1
bond would then be unreactive towards exchange with the N-terminus, just as is the case when an 8-membered S1
ring forms in the Cys2
mutant. If we imagine that cystine could be reduced using the pathway shown in , a mixed Se2
bond would form between enzyme and substrate and this has the reverse context compared to a S1
bond, making exchange very slow. Moreover, if this were the case, the thiolate of CysIC
would have to attack a S atom of cysteine, which compared to the Se atom of selenite, is not very electrophilic. Our proposed mechanisms explain why these Se-containing substrates are largely independent of the presence of the C-terminal Sec residue.
Leaving group pKa
as a determining factor in substrate utilization by mammalian TR is also seen with other known, small molecule substrates such as we have pointed out with DTNB above. This concept explains why GSSG is not a substrate (its leaving group thiol has a relatively high pKa
), while analogues of glutathione such as selenodiglutatione (GS-Se-SG), and S
-nitrosoglutathione are both utilized as substrates by mammalian TR (17
). This is most likely due to the low pKa
of the selenolate in GS-Se-SG and HNO in GSNO (pKa
= 4.7) (41
Disulfide Reductase Activity of TR
A summary of the kinetic data using lipoic acid as a substrate for full-length and truncated TRs is given in (the mitochondrial TR from C. elegans (CeTR2) is included as part of our analysis). As the data in demonstrates, the assumption that Sec is needed to catalyze the reduction of lipoic acid is found to be untrue upon comparison of the full-length WT Sec2-containing enzyme to the full-length Cys2-mutant enzyme as the kcat values are nearly identical. The Km value does increase 2.3-fold in the Cys2 mutant, however. The truncated mammalian enzymes in this study also turned over lipoic acid, consistent with our hypothesis that the reduction of the 1,2-dithiolane ring of lipoic acid is largely due to the reactivity of the N-terminal reaction center. Like our results with small molecule Se-containing substrates above, this result demonstrates again that Sec is not needed to reduce some substrates and the broad substrate specificity is not due to the presence of the Sec residue. However, while the truncated mammalian enzymes would turn over lipoic acid, they did not display saturation kinetics, so we are unable to report a Km value for these mutants. Comparing the activity of mTRΔ8 to the full-length enzyme, we see that this truncated enzyme has higher activity than the WT enzyme, demonstrating that the substrate has greater access to the N-terminal reaction center in the mutant than in the WT enzyme.
Lipoic Acid Reductase Activity of Various full-length and truncated TRsa.
Interestingly, we found that mTRΔ8 had significantly higher activity at pH 6.1. At this pH, mTRΔ8 did show saturation kinetics, with kcat
increasing nearly 3.6-fold compared to the WT enzyme at pH 7.0. The pH rate profiles for mTRΔ8 and the full-length enzyme with lipoic acid as a substrate are shown in . As can be seen in the profile, there is a very sharp drop in activity below pH 6.1 for both enzymes and an explanation for this behavior is currently unknown. A possible explanation is that as the pH becomes lower than 6.0, the thiolate of CysIC
becomes protonated, making thiol/disulfide exchange slow between enzyme and the 1,2 dithiolane of lipoic acid. We previously reported the pKa
as 5.8 (26
). In contrast to the truncated mammalian enzyme, DmTRΔ8 did not show a sharp increase in activity near pH 6 and had a very similar profile to that of its full-length counterpart as shown in . However, if we use lipoamide as a substrate, we see lower activity overall (lower kcat
), but also tighter binding as reflected by a nearly 3-fold drop in Km
(). The pH optimum using lipoamide as a substrate is ~ 8.5 for this truncated enzyme (see Figure S3 in the Supporting Information
). The difference in activities between lipoic acid and lipoamide seems to be the affinity the enzyme has for the negatively charged lipoic acid versus the neutral lipoamide. The data indicates that this affinity is not preferential binding because the neutral substrate has a lower Km
than the charged substrate. We posited that the carboxylate group of lipoic acid was acting as a general acid/general base catalyst. This hypothesis was tested by adding 50 mM acetate to the lipoamide assay buffer solution. The presence of acetate in the reaction buffer resulted in 23.6% higher activity using lipoamide as a substrate at pH 6.1 and 17.5% higher activity at pH 7.0, respectively. This data is consistent with an acid/base catalytic role for the carboxylate group of lipoic acid, but the higher activity could also be the result of a specific binding interaction between substrate and enzyme.
Figure 4 Reduction of lipoic acid as a function of pH. (A) mTRΔ8 (closed squares) and full-length mTR (open squares). The truncated enzyme has an optimum at pH 6.1, while the full-length enzyme has an optimum near 7.5. (B) DmTRΔ8 (closed circles) (more ...)
Lipoamide Reductase Activity of Various Full-length and Truncated TRsa.
The overall results clearly demonstrate that the disulfide bond of lipoic acid/lipoamide is capable of being reduced by the N-terminal redox center and this suggests to us that in the holoenzyme reduction of lipoic acid (and other substrates) can occur via the N-terminal redox center. We speculate that some substrates are in competitive equilibrium with the C-terminal selenosulfide ring for interaction with the N-terminal redox center. This model allows for reduction of small molecule substrates to take place at either site (). This model is similar to the one put forth by Fujiwara and coworkers for the reduction of DTNB by TR (42
). Previously it has been assumed that the reduction of lipoic acid is dependent upon the presence of Sec in the C-terminal redox center (18
Figure 5 Proposed model for the interaction of small molecule substrates with TR. Both the oxidized C-terminal tetrapeptide (Gly-Cys-Sec-Gly) and small molecule disulfide (such as lipoic acid) can bind in the tetrapeptide binding pocket and are thus in competitive (more ...)
Since lipoic acid/lipoamide must also bind in the same place occupied by the C-terminal tail containing either a vicinal disulfide bond in the case of DmTR or a vicinal selenosulfide bond in the case of the mammalian enzyme, the results also show a clear difference in the preference of ring size of various disulfide substrates with each type of enzyme as summarized in and . As shown by the data in , the mammalian enzyme clearly prefers a small-ringed, disulfide substrate. This can be seen in comparing the activities of lipoic acid/lipoamide (5-membered ring) to DTT(ox
) (6-membered ring) and a peptide containing a vicinal disulfide bond (8-membered ring). The mammalian enzyme can utilize an 8-membered ring substrate, but only if a Se atom is present in the substrate. The preference of the mammalian enzyme for the 5-membered 1,2-dithiolane ring of lipoic acid is in spite of the much higher pKa
value for both sulfhydryl groups (10.7) (43
) compared to the 8-membered ring of the peptide containing a vicinal disulfide bond (8.3) (40
). We put forth two reasons for the higher activity of the 1,2-dithiolane ring as a substrate in comparison to the other ring sizes with the truncated mammalian enzyme. First, seminal work by Whitesides and coworkers demonstrated that due to the large ring strain imparted on the 1,2-dithiolane ring system by the highly compressed C-S-S- C dihedral angle (35°), that a 1,2-dithiolane is 650-fold more reactive towards thiol/disulfide exchange reactions compared to a 1,2-dithiane ring (such as DTT(ox)) (46
). Second, the leaving group sulfur atom in this exchange reaction must be positioned to be protonated by the enzymic general acid (His463′). The fact that the 8-membered ring of the peptide containing a Cys1
vicinal disulfide is a very poor substrate could be interpreted as meaning the ring is relatively unstrained in this peptide, or the thiolate of Cys2
is not in the correct position to be stabilized by His463′, either by proton transfer (our previous argument) (26
) or by electrostatic stabilization.
Activity of mTRΔ8 Toward Cyclic and Acyclic Substrates.
Activity of DmTRΔ8 Toward Cyclic and Acyclic Substratesa.
The situation with DmTRΔ8 (a Cys-TR) contrasts with that of the mammalian enzyme as it shows a preference for 8-membered ring
substrates if we compare the same series of disulfides (). However, lipoic acid is still turned over 190-fold faster in comparison to DTT(ox
), but the overall rate is lower in comparison to mTRΔ8. This lower activity with lipoic acid is probably a reflection of the higher pKa
of the attacking thiolate in DmTRΔ8 in comparison to mTRΔ8 (6.5 for Cys57 – CysIC
in DmTR vs. 5.8 for Cys52 – CysIC
in mTR) (26
). If we use the same line of reasoning as above to explain the higher activity of the 8-membered ring of the Cys1
vicinal disulfide peptide substrate in comparison to the other disulfide substrates in , it would mean that there is either a large degree of strain energy in the ring of the enzyme/peptide complex – rendering this peptide substrate highly reactive, or the conformation of the ring allows for correct positioning for the stabilization of the thiolate of Cys2
. We also find that the turnover rate of a homologous peptide in which a Se atom substitutes for a sulfur atom (while still maintaining the size of the ring at 8 atoms) is significantly higher –greater than 2-fold. This fact implies that the presence of a Se atom imparts higher activity to the peptide substrate because of the lower pKa
of a selenolate compared to a thiolate, since Se for S substitution would most likely decrease ring strain as a result of a larger ring size. Both types of truncated enzymes can utilize acyclic substrates if the substrate contains a low pKa
leaving group. This is seen upon comparing the linear, acyclic substrates GSSG and DTNB. The low pKa
leaving group of DTNB compensates for a lack of ring strain as well as the “incorrect” position of the leaving group thiolate relative to reactive groups in the tetrapeptide binding pocket.
Inhibition of TR By Gold Compounds
Conventional wisdom also holds that the reason for the very strong inhibition of TR by various organogold compounds is that the Se atom of the mammalian enzyme makes a very strong coordinate covalent bond with Au (48
). However, it is well known that thiols also have a strong interaction with Au (50
). Given that we have tested several strongly held beliefs about the mammalian enzyme as discussed above, we decided to examine the ability of the mammalian enzyme and the truncated mammalian enzyme to be inhibited by auranofin and aurothioglucose. The results are summarized in and the activity/inhibition curves are given in . The results show that mutation of Sec to Cys causes the IC50
to increase 4-fold in the case of auranofin and 1.7-fold in the case of aurothioglucose, indicating that the Se atom interacts with the Au atom in both of these inhibitors. To further assess the role of the Se atom in binding these inhibitors, we tested the truncated enzymes ability to be inhibited by both compounds. The results show that the binding of auranofin is strongly perturbed by the elimination of the C-terminal tail containing the Gly- Cys1
-Gly tetrapeptide, as the IC50
increases from 75 nM to 850 nM. In the case of aurothioglucose, the truncation mutant shows tighter binding
than the WT enzyme. This demonstrates that a significant portion of the binding interaction of the Au atom of aurothioglucose is with the two thiol groups of the CIC
(N-terminal) active site. We recently demonstrated that the pKa
values of these two thiol groups are: pKaIC
= 5.8 and pKaCT
= 5.02 (26
). Thus, at pH 7, these thiol groups are strongly ionized and can attack the more positively charged Au atom of aurothioglucose (in comparison to auranofin) and form a strong complex. In addition, aurothioglucose is more compact than auranofin and can more easily fit into the tetrapeptide binding site of the enzyme (see for the structures of these compounds).
Inhibition of Mouse Mitochondrial TR and TR Mutants By Gold Compounds.
Figure 6 Inhibition plots of TR using auranofin as an inhibitor (A) or aurothioglucose as an inhibitor (B) for mTRΔ8 (open triangles), mTR-GCCG (open squares), and mTR-GCUG (closed circles). IC50 values are calculated from this plot and reported in (more ...)
Structures of gold containing inhibitors of TR: auranofin (top), aurothioglucose (bottom).
The data in potentially explains some of the differential effects that have been observed with these inhibitors. In a recent study it was shown that when cells were treated with aurothioglucose that there was no increase in the amount of oxidized Trx and there was also no increase in the production of reactive oxygen species (51
). In contrast, cells treated with auranofin underwent apoptosis in a mechanism that depended on pro-apoptotic members of the Bcl-2 family (Bax/Bak
). The auranofin treated cells exhibited signs of mitochondrial oxidative stress such as increased levels of peroxiredoxin 3 (52
). Arnér and coworkers have recently characterized what they have termed as “SecTRAPs” (s
eductase derived a
roteins). A SecTRAP is a form of TR in which the Sec residue has become modified with a cellular electrophile. The modification of the Sec residue apparently unmasks a new function of the enzyme, and it is this new function that causes apoptosis. This group also demonstrated that this new function of TR induced oxidative stress inside the cell and that this activity was dependent upon the CVNVGC active site (53
). Our potential explanation is that auranofin complexes with the Se atom of TR forming a SecTRAP and enhances the function of the N-terminal reaction center and this new function leads to cellular apoptosis, while aurothioglucose completely inhibits the activity of the CVNVGC active site so that the toxic effects are not observed.