Disulfide lnterchange Reactions An Enzymic Case Study Trevw M. Kitson Massey University, Palmerston North, New Zealand This article concerns the reaction known as "disulfide interchange", in whicha thiol reacts with adisulfide togive a different thiol and disulfide pair as the products. Such a reaction is a common occurrence in protein chemistry and enzymology; the specific case of the enzyme aldehyde dehydrogenase is chosen to illustrate various interesting manifestatgns of disulfide interchange. General Equailons for Dlsulflde Interchange In its most general form the equation for disulfide interchange may he written A-S-
-
+ B-S-S-C
A-S-S-B
+ C-S-
(1)
The equation is shown as involving A-Srather than A-SH, as the latter species is of negligible nucleophilicity compared to the former. The reaction will only proceed in the direction shown if C-S- is a better leaving group than A-S- (or, in other words, if, in general, C-SH is a stronger acid than A-SH). Likewise, showing the original disulfide liberating C-S- rather than B-S- implies of course that C-S- is the better leaving group of the two. In practice, the original disulfide is often a symmetrical compound, and eq 1then becomes A-S-
-
+ B-S-S-B
A-S-S-B
+ B-S-
+ A-S-S-B
Assuming sufficient A-Stions 2 and 3 is 2A-S-
A-S-S-A
+ B-S-
(3)
is available the net effect of reac-
+ B-S-S-B
A-S-S-A
+ 2B-S-
(4)
If B-S- is a much better leaving group than A-S- (or, to put it another way, if B-S-S-B has a much larger reduction potential than A-S-S-A), the reaction will proceed essentially to completion with a 21 ratio of reactants as required by t h e stoichiometry of t h e equation; if A-S-S-A and B-S-S-B are of comparable reduction potential, then a large excess of A-S- will ensure that the equilibrium is driven to the right, giving effectively the complete reduction of B-S-S-B. There are very many interesting cases of disulfide interchange in which A-S- represents the thiolate anion of a cysteine residue in a protein. Equations 2 and 3 then become Prot-S-
+ B-S-S--B
-
Prot-S-S-B
+ B-S-
(5)
Sometimes the cysteine residue of interest is on the surface of the protein molecule and reaction 5 proceeds smoothly. Sometimes, however, i t is buried deep within the protein's tertiary structure a n d more or less inaccessible t o B-S-S-B, especially if the latter is bulky; under these circumstances reaction 5 is very slow or completely impossible, unless the protein is first denatured (for example by the use of concentrated urea or guanidinium hydrochloride solu-
-
Prot-S-S-B
\
s-
Prot-S
\/
+ B-S-
(7)
S
Reactions 5 and 7 together convert two cysteine residues to cvstine. Eauation 7 is the intramolecular eouivalent of the intermolecdar equation (eq 6) and again m& not be of high orobahilitv in an undenatured arotein molecule. Having described the generai possibilities, the rest of this article concentrates on saecific examales of disulfide interchange that are exhibited by the enzyme aldehyde dehydrogenase (AldDH). Aldehyde Dehydrogenase and Dlsulfiram This enzyme controls the second step in alcohol metabolism-the oxidation of acetaldehyde to acetate-the conversion of ethanol to acetaldehyde having previously been catalyzed by alcohol dehydrogenase (ADH):
(2)
As soon as any A-S-S-B is formed, i t will tend to compete with B-S-S-B for A-S-, as follows: A-S-
tion). Similarly, reaction 6 is often sterically precluded when dealing with large protein molecules in their native conformations. Many proteins of course have a number of cysteine residues per molecule; this gives rise to a further possible reaction,
Ethanol
-
ADH
Acetaldehyde
-
AldDH
Acetate
(8)
I t has been known since 1948 that disulfiram or "Antahuse" [(CzHs)2N-CS-S-S-CS-N(C2H&] has the ability to block the action of AldDH in the liver. This underlies the use of this drug in alcoholism therapy; the alcoholic patient knows that if (s)he drinks after taking disulfiram (s)he will experience a very unpleasant reaction. The symptoms result from the greater than usual concentration of acetaldehvde that build; up in the blood under these circumstances (i). Originallv it was thought that disulfiram interferes with the action of the enzyme by binding to i t as a noncovalent inhibitor (2).However, the compound has no structural similarity to the enzyme's substrates (aldehydes and NAD+), and later studies using AldDH isolated from sheep liver (3) supported disulfide interchange as the mechanism for the action of disulfiram. For example, after inactivation of the enzyme by treatment with disulfiram, prolonged dialysis does not restore the activity (as would have been expected for a noncovalent inhibitor). A twofold excess of 2-merca~. toethanol immediately destroys disulfiram [according to eq 41, hut it does not reverse the loss of artivity caused by prior mixing of enzyme and disulfiram; again this is inconsistent with reversible noncovalent inhibition, Thus bv 1978 it was considered that this is how disulfiram and AMDHreact: Enz-S-
+ EtzN-CS-S-S-CS-NEtz
-
Em-S-S-CS-NEt2
+ Et2NCS2-
(9)
The reaction is very fast and specific. Onlv a small number (1 012) of the 44 thioigroups in ;he AldDH molecule have to be modified to remove most (93';) of the enzyme acti\,its. Further progress came in 1982 when an attempt was made Volume 65
Number 9 Se~tember1988
829
to tag the enzyme with 14C-labelled disulfiram (with the intention of then digesting the protein, isolating the labelled peptide, and identifying it) (4). Surprisingly, after treatment of AldDH with a low concentration of 14C-disulfiram, followed by prolonged dialysis, there was found to be no radioactivity associated with the protein. Accordingly, i t was concluded that reaction 9 is followed by reaction 10 [a specific case of eq 71:
\
s- (8)
2.2'-dithidi*yridi..e
/
s- (A)
/
\I s
Enz \
Subsequently, using rapid small-scale gel filtration to remove unbound radioactivity, i t was shown that the initial disulfide interchanee that labels the enzvme is vervfast and is responsible for &e observed loss of enzyme aciivity and that the second disulfide interchanee (leadine to the formation of an internal disulfide in the enzyme) is relatively slow, with a half-life of about 12 min a t 25 OC (5).The disulfide formof theenzyme ran beslowly reduced back to itsoriginal enzymatically active form by a hiah concentration of 2-mercaptoethanol or dithiothreitol [effectively according to eq 4 above], but not by the bulkier, naturally occurring reducing agent, glutathione (y-L-glutamyl-L-cysteinylglycine).Presumably this explains why in vivo reversal of the inactivation brought about by disulfiram is relatively slow and dependent on the synthesis of fresh enzyme. Disulfiram is not the only compound with which AldDH undergoes disulfide interchange as will now he described. Aldehyde Dehydrogenase and 2,2'-Dithlodlpyrldlne The 2,2'- and 4,4'-isomers of dithiodipyridine have been extensively used for the modification and estimation of protein thiol groups (6).Any expectation that 2,2'-dithiodipyridine would inactivate AldDH just as disulfiram does was quickly dispelled, however, hy the surprising observation that when enzyme freshly modified by 2,2'-dithiodipyridine was assayed in the usual way it was about twice as active as the native enzyme. Furthermore, this activated enzyme was immune to the inactivatory effect of disulfiram (7). An advantage that 2,2'-dithiodipyridine has over disulfiram is that the reaction product is a chromophore, and this was put to use in the following experiment. Enzyme labelled by reaction with 2,2'-dithiodipyridine was quickly isolated of 2by gel filtration and observed a t 343 nm (the,,A thiopyridone). This showed that, a t 25 'C, 2-thiopyridone was liberated from the labelled enzyme in a first-order reaction with a half-life of approximately 3 h, while simultaneously the enzyme activity gradually disappeared (8). I t was concluded, therefore, that both disulfiram and 2,2'-dithiodipyridine end up by converting AldDH to the same internal disulfide form, but a t this stage the order of modification of the two thiol groups involved (termed "A" and "B") was not known. That is, either eq 11or eq 12 could apply:
S-S-2-pyridyl
(12)
One might consider eq 11is simpler, in which a particularly reactive thiol group ("A") is specifically modified first by either reagent. However, this would mean that group "A" could not play the role of the essential nucleophile that is often assumed to take part in the mechanism of the reaction that AldDH catalyses (9).(If group "A" were essential, then any modification of it must of course result in inactivation.) On the other hand, eq 12 maintains the attractive hypothesis that group "A" is the catalytically essential group. In the case of 2,2'-dithiodipyridine, only when this group is modified by the second, intramolecular, disulfide interchange reaction does the enzyme activity fall. I t is easier to see from eq 12 than from eq 11why the intermediate labelled forms of the enzyme should be so different in activity (one inactive, one activated) since in the second case different enzymic residues are involved. However, the protection against disulfiram that is brought ahout by modification with 2,2'-dithiodi~vridinemust in ea 12 be due to the steric blockine of ar& of disulfiram a i d not directly due to the prior moiification of the critical arour, ("A") as in ea 11. Evidence to help-dec& between t6e two possihilities shown in eqs 11 and 12 was sought using unsymmetrical analogues of the symmetrical compounds disulfiram and 22-dithiodipyridine, as described in the next section. Aldehyde Dehydrogenase and Mixed Methyl Dlsulfldes T h e effect of methyl diethylthiocarbamyl disulfide (EtzNCS-S-S-Me) and methyl 2-pyridyl disulfide ([C5H4N]-S-S-Me) on AldDH was investigated. I t was reasoned that both these reagents would label the enzyme with the -S-Me group, since in both cases the other moiety involved (diethyldithiocarbamate and the anion of 2thiopyridone, respectively) is a much better leaving group than the methanetbiolate ion. The reactions involved are simply versions of eq 1: Enz -S-
+ EtzNCS-S-S-Me
-
The synthesis that was used for these reagents is itself another example of the disulfide interchange reaction, utilizing the commercially available methyl methanethiosulfonate (10):
Enz \
, ,Enz I SS-2-pyridyl
S- (R) 2,2,-diLhidipvridi..
/
\
S
Em \
830
Journal of Chemical Education
(11)
(Again it is the anion expected to be the better leaving group that is indeed released from the unsvmmetrical reaeent.) It wan found that methyl diethy~thiocarbam~l d i h f i d e is a potent inactivator of AldDH iust as disulfiram is. whereas when methyl 2-pyridyldisulfide reacts with the enzyme (and
the course of reaction was confirmed bv observation of the release of 2-thiopyridone) the activity i s slightly increased (11). The fact that different reagents supplying the same label have different effects on the enzymic activity proves that different enzvmic residues are involved here. The alternative idea that 'the enzyme has a particular thiol group uniquely reactive to any modifier becomes untenable. This in turn supports eq 12 for the way in which the corresponding symmetrical reagents react. The potent inactivation caused by modifying group "A" with a label as small as -S-Me (from Et2NCS-S-S-Me) is consistent with the proposal that this group is essential. Interestingly, when the enzyme is labelled by 2.2'-dithiodipyridine a t group "B" it is protected against the reaction of disulfiram a t group "A" (as mentioned above) but not against the smaller methyl diethylthiocarhamyl disulfide. In the next two sections, questions concerning the action of disulfiram on AldDH in vivo are addressed. Dlsulflram and Glutathlone One problem is that upon absorption into the bloodstream disulfiram becomes raoidlv and ouantitativelv reduced t o diethyldithiocarbamat; hy"g1utatkone as folloks (12): EhNCS-S-S-CSNEt2
+ 2G-S-
-
GS-S-G
+ 2EtzNCS2-
(16)
This is a version of the general equation (eq 4); G- stands for
Administration of diethyldithiocarbamate inactivates AldDH in vivo hut has no effect on the enzyme in vitro. I t seems therefore that in the liver there must be, at least to some extent, reoxidation of diethyldithiocarbamate to disulfiram in order for inactivation of the enzyme to occur. Several agents have been reported to be capable of bringing about this oxidation. such as cvtochrome c. methaemoelobin. xanthine oxidase,and cataiase (13).~xperimentsin vitru'have shown that AldDH comoetes well aeainst excess elutatbione for low concentrations of disulfiram (14) [that is, reaction 9 is much faster than reaction 161 and mesumablv a similar situation applies in vivo even though-the concentration of glutathione is as high as 5mM. Mltochondrlal Aldehyde Dehydrogenase All the results referred to so far have been obtained with cytoplasmic AldDH. Broadly speaking though, human liver and most other types of mammalian liver (except rat) contain roughly equal levels of AldDH activity in the cytoplasmic and mitochondrial fractions of the cell. The cytoplasmic enzyme is very sensitive in vitro to rapid inactivation by low concentrations of disulfiram; the mitochondrial enzyme is virtually unaffected. However, it is considered that the major proportion of acetaldehyde oxidation during alcohol metabolism occurs in the mitochondria of the liver cell (151. This leads us to another problem concerning the action of disulfiram i n v i v e f o r a reaction against alcohol to be manifested, mitochondrial AldDH should be inactivated, but paradoxically i t is this form that is immune to disulfiram. Recently a solution to this dilemma has been proposed (16). It involves not so much the reoxidation of diethyldithiocarbamate to disulfiram itself (although this might still occur), hut rather the cooxidation of diethyldithiocarbamate and endoeenousmethanethiol toeivemetbvldietbvlthiocarbamyl dihfide. Unlike disulfirah, the m&ed methyl disulfide is a potent inactivator not only of the cytoplasmic but also of the mitochondrial form of AldDH. The site of reaction in the mitochondrial enzyme appears to be sterically
accessihle to the smaller unsymmetrical reagent but not to disulfiram irself. This is reminiscent of the wav in which the cytoplasmic enzyme, when premodified by 2,~'dithiodipyridine, is also insensitive to disulfiram but not to methyl diethylthiocarhamyl disulfide. Identity of the Reactlve Cystelne Residues The complete primary structure of both forms of human liver AldDH has been determined (17). There is indirect evidence pointing to Cys-302 as being the one that first reacts with disulfiram (the erouo called "A" above). Interestingly in the cytoplasmic enzyme residue 301 is also cysteine, and in the mitochondrial form residues 301,302, and 303 are all cysteine (a rare sequence in protein primary structure). However, it is thought that the other cysteine ("B") that becomes linked to "A" as the end result of treatment with disulfiram is probably yet another (so far unidentified) residue, situated on some other part of the polypeptide chain that so happens to be folded close enough for reaction 10 to occur. Aldehyde Dehydrogenase and Cephalosporln Antlblotlcs In the last few years certain cephalosporin antibiotics such as moxalactam, cefamandole. and cefooerazone have been found to induce an unple&ant diskfiramlike reaction against alcohol. These comoounds all vmsess a l-methvlretkole-5-thio side chain. he claim (18) that they arenomovalent inhibitors of AldDH in vivo is unlikely to he correct because (1)the inhibition in vitro is very weak, (2) similar compounds with a different side chain have no effect on alcohol metabolism. and (3) the free l-methvltetrazole-5thiol behaves like the pardnt antibiotics in vice but has no effect on AldDH in vitro. Instead it bas been orooosed (19) that the side chain becomes liberated in the body and bx; dized either to the svmmetrical 5.5'-dithiobidl-methvltetrazole) or to the unsy&metrical methyl 6-(l-m&hyltet~azolyl) disulfide. These comoounds could then react with AMDH by the familiar disuifide interchange reaction:
The symmetrical disulfide was found to inactivate the cytoplasmic enzyme in vitro in a similar way to disulfiram (191, whereas recent experiments (20) have shown that only the mixed m e t h y l d i s u l f i d e (like i t s analogue, Et2NCS-S-S-Me) is a potent inactivator of both the cytoplasmic and mitochondrial forms of AldDH in vitro. Thus the Volume 65 Number 9
September 1988
831
story of what may he involved in the effect of cephalosporins on alcohol metabolism has followed a parallel development to that of disulfiram-from noncovalent inhibitor, to symmetrical disulfide, to mixed methyl disulfide. Conclusion
Aldehyde dehydrogenase has proved to be a particularly instructive case to study in connection with disulfide interchange reactions. The enzyme reacts with a number of disulfides with widely differing effects on enzymic The reactions show interesting steric effects, and in some cases a second interchange reaction leading to internal disulfide formation follows the initial labelling reaction. Valuable information has emerged from these studies about the antago,,ism to alcohol induced by certain drugs, and in connection with the enzyme's possible mechanism of action.
832
Journal of Chemical Education
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'2. Cabby, J.: Msyersohn, M.; Selliah, S. J. Phormoroi. EX^. T ~ S T .1977,202, 724-731. . 13. Inouye, K.:Fukunaga, M.: Yamasawa, K. Life S C ~1982,30,419-424. 14. k its,.,^. ~.a,h,,. J 1gs1.199,25~.258. 15- P="h. R.: Ohkma. K.: Lindros. K. 0.; Zirnmerman. U.-1.P.; Kabayashi, K.: WilliamJ0n.J. R. J.Biol. Chem. 1914,249,49264933. 16. MaeKem1l.A. 0.: Va1lari.R. c . : P ~ ~ ~ ~ u ~ z ~ ~ , R .1985,179,77-81. FEBsL~LL. 17. Hernwl, J.:Ksiser,R:~ornvsll, H. E U ~ ~. . ~ i o r h e m 1m.153,13-28. . 1 8 F'ewdt. K.J.: Schreiner. E.: Chri8tmsnn~Kleisa.U.Inlection 1985.13,91. 19. Kitson, T M. Alcoholism Clin. Exp. Re'. 1988.1O. 2732. 20. ~ o o r n e s . ~ . ~ its^^,^. .; M., v n p u b ~ i a Fh~~S U I ~ .
