Influence of Tertiary Structure on Nucleophilic Substitution Reactions

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Chem. Res. Toxicol. 1996, 9, 918-923

Perspective Influence of Tertiary Structure on Nucleophilic Substitution Reactions of Proteins Paul L. Skipper Division of Toxicology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139 Received February 19, 1996

Introduction Electrophilic compounds undergo nucleophilic substitution reactions with proteins as a natural consequence of the nucleophilic character of over one-half of the common amino acid side chains. This reaction to form adducts is a focus of various research interests, including mapping drug binding sites through affinity labeling, characterizing mechanism-based enzyme inactivation, as well as carcinogen dosimetry. In all these areas, there has been an interest in rationalizing product control. A variety of factors have been considered, including chemical reactivity, steric accessibility, and active site proximity. None of these has been broadly successful in explaining why particular amino acids are targeted for reaction; clearly, additional factors are involved. Reaction of electrophiles with another prominent biological macromolecule, DNA, has similarly challenged researchers to explain the forces that govern which products prevail. Here, the easily visualized concept of intercalation (1, 2), as an event that precedes covalent bond formation, has proved useful. It has been demonstrated that planar electrophiles such as aflatoxin B1 (AFB1)1 epoxide (3) and some polycyclic aromatic hydrocarbon (PAH) diol epoxides (4, 5) are bound between two stacked base pairs in double-stranded DNA before they form a covalent bond. Since double-stranded DNA has a high degree of order, it has even been possible to demonstrate how intercalation can orient the electrophile for reaction with a specific nucleophilic center (6). Most proteins have no regular and well-defined structural motif like the stacking of bases in the DNA helix. Nevertheless, the directing influence of intercalation is a concept that can be carried over to the protein reaction. Intercalation is, after all, nothing more than a highly specialized case of receptor-ligand type binding. It is well understood that noncovalent interaction energy for the binding of ligands by proteins can be great enough to produce equilibrium association constants up to 1012 M-1. What may not be fully appreciated is that these large equilibrium constants are driven by association kinetics that are extremely fast compared to nucleophilic substitution reactions. Thus, in many cases, noncovalent binding may be kinetically highly favored over unassisted nucleophilic substitution reactions. In this article, it is proposed that “docking” of an electrophile in a receptor region of a protein, as il1Abbreviations: AFB , aflatoxin B ; BPDE, r-7,t-8-dihydroxy-t-9,t1 1 10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene; COHb, carbonmonoxyhemoglobin; p-CMB, p-(chloromercuri)benzoate; ECA, ethacrynic acid; EMS, ethyl methanesulfonate; Hb, hemoglobin; PAH, polycyclic aromatic hydrocarbon; TIB, 2,3,5-triiodobenzoic acid.

S0893-228x(96)00028-8 CCC: $12.00

Figure 1. A kinetic scheme for reaction of small electrophiles with proteins. ka and k-a are the rate constants for reversible, noncovalent protein-electrophile binding. The ratio of ka to k-a defines the equilibrium association constant. k1 is the rate constant for reaction of an electrophile E with a surfaceaccessible nucleophile N1. It is assumed that k1 is not substantially different from the rate constant that characterizes the same reaction with a free amino acid or any other small molecule that has N1 as part of its structure. k2 is the rate constant for covalent bond formation between E and nucleophile N2, which is located within the binding site for E. Some values for ka, k-a, and k1 have been measured or estimated, but values for k2 are largely unknown.

lustrated in Figure 1, is generally the first step on the path to covalent adduct formation, and as a result, only appropriately situated nucleophiles within the receptor are available for reaction. The kinetic basis for this proposal forms the first part of the discussion. The minimal scheme of Figure 1 is, of course, a highly simplified view of actual events. Reaction at nucleophilic centers outside of well-defined receptor regions may involve some degree of preliminary association as well, and there may exist other competing pathways such as ligand binding in a receptor that fails to result in the formation of covalent adducts. Nevertheless, the model proposed here begins to rationalize a variety of previous results that are not otherwise explicable. Interpretation of some of these results in terms of precovalent binding forms the basis for the latter part of the article.

Reaction Rates and Ligand Binding Rates In the two-step model proposed here, it is necessary that the protein has one or more receptors, or binding sites. Two proteins that have been investigated in some detail are serum albumin and hemoglobin (Hb). The requirement that a protein have binding sites is easily satisfied in the case of serum albumin. This protein readily binds a wide variety of ligands, with a particular affinity for anionic compounds (7, 8). Fatty acids are bound with association constants on the order of 107 M-1. Binding of numerous other compounds has been determined, including many that are nonionic. Association constants vary widely, from relatively weak up to the © 1996 American Chemical Society

Perspective

strength of those displayed by the fatty acids. The ability of hemoglobin to bind small molecules as ligands is less well understood. The most extensive information comes from the study of anti-sickling drugs (9), whose association constants range up to about 103 M-1. Hemoglobin does not appear to have any high affinity binding sites. Enzymes, as well as other proteins with substrate-specific receptors, may also bind nonsubstrates (see below), but this is a less well explored area. The equilibrium association constant for proteinligand binding does not of itself characterize the association rate constant since it is a function of both the forward rate constant and the reverse rate constant. Nevertheless, consideration of a range of measurements that have been made of ligand association rates leads to the conclusion that the equilibrium association constant is influenced predominantly by the dissociation rate constant. Association rate constants vary over a smaller range, so a representative value or small range of values is reasonable to use in the following analysis. Kinetics of the antibody-antigen interaction have been studied in considerable detail (10, 11). Association rate constants on the order of 106-108 M-1 s-1 are typical, while dissociation rate constants vary between about 10-4 and 101 s-1. These values correspond to association constants of 1010-106 M-1. The extremely high affinity Ah receptor binds dioxin with a forward rate constant (107 M-1 s-1) that is in the same range as the antibody constants, even though its equilibrium association constant is greater than 1011 M-1 (12). It is not obvious whether the association rate constant would also have a comparable value for much lower affinity binding such as that observed for hemoglobin and the anti-sickling drugs. Intercalation of benzo[a]pyrene diol epoxide (BPDE) into DNA may be a useful gauge since this intercalation is characterized by an association constant of about 103 M-1 (4), which is of comparable magnitude. The association rate for intercalation of BPDE was not determined exactly, but the binding was described as occurring on the millisecond time scale. Under the reaction conditions used (ca. 10 µM BPDE, 1 mg/mL DNA), the initial velocity of binding can be approximated as 1 µM/ms, or 10-3 M s-1. Dividing this rate by the concentrations of BPDE and DNA (defined as concentration of base pairs, since it is assumed that each base pair represents an intercalation site) yields a value of approximately 105 M-1 s-1 for the second-order rate constant. This derived value is actually quite similar to the rate constants that characterize the high affinity binding discussed above and suggests that low affinity protein binding will exhibit similarly rapid association kinetics. The binding of oleate and N-acetyltryptophan to serum albumin are characterized by a forward rate constant greater than 106 M-1 s-1 (13). Although the equilibrium constants in each case are characteristic of high affinity binding, the initial binding reaction appears to occur at a low affinity binding site. High affinity binding results from a restructuring of the initially formed complex. These experiments thus provide further evidence that ligand-protein association is likely to be very rapid, regardless of the strength of the equilibrium binding. Covalent bond-forming reaction rate constants may be compared to these ligand binding association rate constants. Rate constants have been reported for the reaction of ethyl methanesulfonate (EMS) as well as ethylene oxide with hemoglobin. These constants are probably good approximations to the true rate constants

Chem. Res. Toxicol., Vol. 9, No. 6, 1996 919

Figure 2. Structures of compounds referred to by nondescriptive name in the text. 1, Aflatoxin B1 epoxide; 2, ethacrynic acid; 3, symetryn sulfoxide; 4, tolmetin glucuronide.

for the reactions as they would occur if the protein structure exerted no influence on the reactions, since the interactions between EMS or ethylene oxide with hemoglobin are likely to be small. The values of 1.5 × 10-4 L (g of Hb)-1 h-1 for EMS (14) and 1.8 × 10-4 L (g of Hb)-1 h-1 for ethylene oxide (15) correspond, respectively, to 2.7 × 10-3 and 3.3 × 10-3 M-1 s-1. These values are 8-10 orders of magnitude less than the lowest estimate made earlier of the rate constant for protein-ligand association. The comparison may not be entirely relevant: ethyl methanesulfonate and ethylene oxide do not exhibit the high degree of reactivity typical of many carcinogen metabolites such as PAH diol epoxides. Unfortunately, there is little kinetic data available for these compounds. Rate constants for the reactions with the individual nucleophiles that are part of protein structure have not been reported, nor are there data for reaction with proteins, but these would be of limited usefulness in any case since the reaction profiles are likely to be different than those for reaction with individual nucleophilic amino acids. Estimates for k1 can be obtained, though, from the Swain-Scott equation (16) since solvolysis (H2O) data do exist. BPDE has been reported to undergo hydrolysis in buffer at pH 7.2 with a pseudo-first-order rate constant of 1.2 × 10-3 s-1 (17). Taking 55 M as the concentration of H2O in this experiment, the corresponding secondorder rate constant is 2.2 × 10-5 M-1 s-1. Data also exist for the hydrolysis of AFB1 epoxide (1, Figure 2), a metabolite that is considered to be one of the most reactive. The exo epoxide solvolyzes in 1:1 acetone/ sodium phosphate buffer (pH 7.0) at 23 °C with a rate constant of 1.6 × 10-2 s-1 (6). Under physiological conditions, this value would be severalfold higher, or approximately 10-1 s-1, and the corresponding secondorder rate constant would be approximately 10-3 M-1 s-1. A nucleophilic constant is required for solution of the Swain-Scott equation. Data exist for both the hydrolysis of EMS (18) and its reaction with the protein hemoglobin (14). A composite nucleophilic constant for hemoglobin can thus be derived:

log(k/k0) ) SEMS × N k0(EMS + H2O) ) 2.9 × 10-7

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k(EMS + Hb) ) 2.7 × 10-3 assuming the concentration of nucleophilic centers available for reaction is 50-fold greater than [Hb]; then

EMS + Nu ) 5.4 × 10-5 therefore

k/k0 ) 2.3 and N ) 2.3/SEMS The electrophilic constant, SEMS, has not been determined, but it is likely to be similar to the constant for ethyl tosylate, which is 0.67 (19). In that case, the value for N will be 3.4, which lies in the middle of the range of values determined for the nucleophilic groups present in proteins. Using the derived value of N, and a range of values for S (since these are not known), rate constants that would characterize the reactions of BPDE and AFB1 epoxide with proteins if the proteins behaved simply as a localized concentration of nucleophilic centers (k1 in Figure 1) can be estimated. These range from 0.015 to 2.9 M-1 s-1 for BPDE and from 0.7 to 130 M-1 s-1 for AFB1 epoxide when (0.5)SEMS e S e (1.5)SEMS if it is again assumed that there are 50 nucleophilic centers per mole of protein. Carboxylate, imidazole, and amino groups are the principal contributors to the nucleophilic constant 3.4 used in these calculations, which is not representative of the thiolate group. Its nucleophilicity is 3-4 orders of magnitude greater so the reaction between cysteine and an electrophile with high S could occur at rates comparable to association rates if the cysteine existed predominantly in the ionized form. The contribution of this special case to adduct formation must be assessed with consideration of the infrequent occurrence of the reduced form of cysteine in most proteins. In summary, it appears that association of a ligand with a protein occurs with a rate constant on the order of 106 M-1 s-1 or greater if there exists in the protein a binding site of even moderate affinity. Except in special cases, reactive electrophiles can only undergo nucleophilic substitution with a rate constant up to about 10 M-1 s-1. There is clearly significant uncertainty in these estimates, but the difference between the two types of rates is so large that the general conclusion is inescapable: nucleophilic substitution reactions of surface-accessible nucleophiles are not competitive with ligand association. The role that preliminary association plays in determining k2, the rate constant for nucleophilic substitution that takes place subsequent to noncovalent association, is an unexplored field, but as long as k2 is not very much less than k1, then preliminary association should cause reaction with N2 to predominate over reaction with other residues.

Hemoglobin Adducts The role of precovalent binding in the formation of hemoglobin adducts was indicated as early as 1986 by Perutz et al. (9) in an investigation of stereochemistry of binding of anti-sickling drugs to human hemoglobin. One of the compounds examined, ethacrynic acid (ECA, 2, Figure 2), is capable of forming a covalent bond through Michael addition to its R,β-unsaturated ketone functionality. It was found that ECA bound irreversibly to hemoglobin at two sites. One was the expected Cys F9(93)β residue: this residue is generally recognized as the most reactive center in human hemoglobin. ECA also

Skipper

reacted with His G19(117)β. This reaction proceeded with very different occupancies at the two sites (β1, β2), which are related by molecular symmetry but are not related crystallographically. Perutz left no doubt in this paper that he regarded this remarkable selectivity to be the result of precovalent binding. Perutz also noted that other anti-sickling drugs were bound in (different) regions of the protein that were believed to be occupied by close-packed amino acid side chains and induced distortions in the normal structure. A similar result was observed in the crystallographic structure of hemoglobin that had reacted with nitrosobiphenyl to form an adduct at Cys F9(93)β (20). Thus, apparent absence of space in the native structure in which to fit a reactant molecule should not necessarily be taken to mean that the molecule cannot be accommodated. As discussed later, though, there may be a limit to the size of ligands that can be accommodated. A second instance of highly specific adduct formation between human hemoglobin and an electrophile is the reaction of BPDE with Asp CE5(47)R (21). The stereochemistry of this interaction is unknown; the reactive amino acid residue was determined not by crystallography but rather by identification of a modified tryptic peptide. This indirect approach leaves open the possibility that while Asp CE5(47)R is not the only residue that reacts to a significant extent, it is the only one to yield a stable product. The question arises becauses the Asp(47) adduct itself is unstable in the nontetrameric form of hemoglobin. It is possible that the ester formed by alkylation of Asp CE5(47)R is stabilized by being situated within a hydrophobic pocket while other esters, although formed, are hydrolytically unstable. This possibility seems remote, though: it would require that not one of the remaining 54 (or 55, if Asp CE5(47)R (R1) or Asp CE5(47)R (R2) exhibits differential reactivity) is similarly situated. Studies of the alkylation of rat hemoglobin are also revealing. Rat hemoglobin possesses a reactive β-chain cysteine in position 125 that is not present in human hemoglobin. It has been shown (22) that this residue is the site of alkylation by the s-triazene herbicide metabolite symetryn sulfoxide (3, Figure 2), as well as by diol epoxide metabolites of fluoranthene (23). The expected Cys F9(93)β is not alkylated to a significant degree by these two electrophiles. In the case of symetryn sulfoxide the selectivity is not the result of differential reaction rates because extended reaction with excess sulfoxide failed to yield any 93β adduct. The failure of these two electrophiles to react at Cys F9(93)β can instead be ascribed to steric inaccessibility, given current understanding of the structure of human hemoglobin. The 125β cysteine, in contrast, is a surface residue and is presumably quite accessible. Nevertheless, when steric constraints are absent, the cysteine in position 93 is the more reactive: kinetic studies with p-(chloromercuri)benzoate (p-CMB) indicate that this sulfyhydryl-specific reagent reacts 50-fold faster with Cys 93β than with Cys 125β (24). Thus, greater accessibility is not the determinant of reactivity. Even the most rapid reaction of p-CMB with a hemoglobin sulfyhydryl has a rate constant of about 6 × 105 M-1 s-1, which is less than ligand association rate constant estimates made earlier. It is thus quite reasonable to attribute the high reactivity of Cys F9(93)β with p-CMB to precovalent binding.

Perspective

Chem. Res. Toxicol., Vol. 9, No. 6, 1996 921

The reaction of nitrosoarenes with hemoglobin also needs to be considered in the interest of completeness, even though the results are consistent with more than one interpretation. Nitrosoarenes such as nitrosobenzene, numerous nitrosoalkylbenzenes, nitrosobiphenyl, etc., react with hemoglobin by addition of a sulfhydryl -SH across the NdO bond (25). The initial reaction product rearranges to the more stable sulfinamide structure (26). Initial rate constants for the reaction of nitrosobenzene and several substituted nitrosobenzenes with carbonmonoxyhemoglobin (COHb) have been measured; they fall in the range 1-10 M-1 s-1 at 25 °C, pH 7.4 (27). In contrast, the reaction of nitrosobenzene with glutathione occurs with a rate constant nearly 100-fold greater (28). The difference in rate constants suggests that intraerythrocytic glutathione, which is present in roughly the same molar concentration as hemoglobin, should effectively prevent formation of hemoglobin adducts, yet it has been shown that glutathione and hemoglobin compete almost equally for reaction with nitrosobiphenyl in vitro (29). One explanation for the parity of product yields in the competition between glutathione and hemoglobin for nitrosobiphenyl could be that there exist binding sites in hemoglobin, other than the pocket around Cys F9(93)β, in which nitrosobiphenyl would be sequestered rapidly and thereby prevented from reacting with glutathione. Binding in those other locations would not lead to other adducts since nitrosoarenes undergo no reaction with COHb pretreated with maleimide. Similarly, complexation with heme iron could serve the same function. This reaction is considerably more rapid than the reaction of nitrosobenzene with glutathione (k ) 5 × 103 M-1 s-1), taking place with a rate constant on the order of 105 M-1 s-1 (30). It would then only be necessary that the bound nitrosoarene could migrate from the initial binding site to the cysteine without encountering the glutathione present, that is to say, without becoming a free solute.

Serum Albumin One of the earliest indications that serum albumin covalently binds the electrophilic products of carcinogen metabolism site-selectively was the identification of a tryptophan adduct as the product of reaction of human albumin with N-(sulfonyloxy)-N-acetyl-4-aminobiphenyl (31). The human protein contains only one tryptophan residue, which is not accessible from the surface, so this seemed an improbable locus for reaction on the basis of abundance and reactivity. In agreement with the anticipated low reactivity of tryptophan toward the sulfate ester, attempts to obtain reaction with free tryptophan were unsuccessful.2 Nevertheless, it was later shown that analogous tryptophan adducts were also the products of reaction of human serum albumin with the hydroxamic acid sulfate esters derived from the structurally similar aromatic amines, 2-aminofluorene and benzidine (32). At the time this work was performed, accurate molecular models of serum albumin were unavailable, so it was difficult to determine if there was a stereochemical basis for the high selectivity of the hydroxamic acid sulfate esters for the tryptophan residue. Since then, an X-ray crystallographic structure of albumin has been determined (33), which makes it possible to begin to under2G.

Bu¨chi, personal communication.

stand the forces controlling this reaction. Unfortunately, the atomic coordinates have not been made available, so the interpretation that follows is based on limited information. Tryptophan 214 forms part of a binding site in serum albumin designated IIA, which has affinity for a wide range of ligands. One of these is 2,3,5-triiodobenzoic acid (TIB) which was used in cocrystallization studies to map the binding site. Its interactions with various amino acids that make up the binding site may be relevant to understanding the binding of the hydroxamic acid sulfate esters. Thus, both types of ligands are characterized by a negatively charged functional group located at one end of an extended region of hydrophobicity. Binding site IIA is complementary, with a large hydrophobic pocket at one end and several positively charged amino acid side chains at the other. It appears, from the published images of this binding site, that if the aromatic amine metabolites were bound in this site in the same orientation as TIB that they would be suitably positioned to react with the indole ring of the tryptophan residue. Stereochemical control of the reaction between a carcinogen and albumin has been demonstrated in albumin adduct formation by BPDE (34). When albumin is treated with the (-) enantiomer, the principal product results from alkylation of the imidiazole ring of His146 by the diol epoxide. None of this alkylhistidine is formed by the (+) enantiomer; instead, the reaction proceeds via esterification of Asp187 or Glu188. Since albumin exists as a single enantiomer, such selectivity is not surprising. Again, with reference to the crystal structure of the protein, it is possible to begin to understand the origins of this selectivity. His146 and Asp187/Glu188 are apparently quite close to each other in the native structure as elements of the IB subdomain of albumin. This much is fairly certain from inspection of the published images. Thus, it appears that the product difference does not arise as the result of precovalent binding in different regions of the protein, but by reaction with different nucleophiles within the same binding site. This inference is supported by other studies that demonstrate broad specificity of the IB binding site for PAH diol epoxides: definitive evidence has also been presented for alkylation of His146 by 3,4dihydrobenz[a]anthracene-3,4-diol 1,2-epoxide, 8,9-dihydrobenz[a]anthracene-8,9-diol 10,11-epoxide, and 9,10dihydrobenzo[b]fluoranthene-9,10-diol 11,12-epoxide along with tentative identification of adducts formed by 2,3dihydrofluoranthene-2,3-diol 1,10b-epoxide and 9,10-dihydrobenzo[a]pyrene-9,10-diol 7,8-epoxide (35-37). Interestingly, 10,11-dihydrodibenz[a,c]anthracene-10,11diol 12,13-epoxide does not appear to alkylate this histidine. Except in the case of BPDE, reaction with carboxylic side chains has not yet been determined. The enantioselectivity exhibited by BPDE might arise if the orientation of the aromatic ring system within the binding site was similar for each enantiomer and the nucleophile that opens the oxirane ring attacked in the same way in each case (backside or frontside attack). Although this seems an attractive mechanism, others are equally plausible. Better understanding of the docking involved must await the availability of the coordinates of the crystal structure of albumin. The glucuronide metabolite of the drug tolmetin (4, Figure 2) also binds site-specifically to serum albumin (38), and its interactions with the protein are readily understandable in terms of the molecular model of the

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protein described by He and Carter (33). Tolmetin glucuronide reacts predominantly with lysines 195 and 199 (ca. 60%). Lysine 199 is one of the positively charged amino acids that form the focal point for orienting the anionic portion of subdomain IIA ligands. The tolmetin half of tolmetin glucuronide appears to be relatively hydrophobic, while the glucuronic acid part carries a negative charge. Since it is the anomeric carbon of the glucuronic acid that is the electrophilic center of reaction (after acyl migration to one of the hydroxyl groups), it is not surprising that reaction occurs at the lysines that orient the molecule.

important consequence of precovalent binding being a common occurrence is that predictions regarding likely target sites will require far more sophisticated methods than are often applied today. In particular, good molecular models of the proteins of interest will be essential. Recent progress is encouraging. In the last several years the crystallographic structures of both the histone octamer (44, 45) and serum albumin (33) have been determined. With the advent of increasingly powerful methods for calculating interaction energies, it may soon become possible to model the relevant precovalent binding.

Other Proteins

Acknowledgment. Financial support was provided by Grants ES04675 and ES05622 from the National Institutes of Health. The author also wishes to thank Billy W. Day for many helpful discussions, especially those regarding the structure of serum albumin.

The study of protein-carcinogen adducts originated with the observations made nearly half a century ago that aminoazo dyes bound irreversibly to rat liver proteins (39). Prior to the advent of modern analytical instrumentation, it was shown that this binding occured by alkylation of methionine, as inferred by isolation of methyl thioethers upon vigorous hydrolysis of the dyebound proteins (40, 41). More recently, the specificity of binding by one member of this class, 3′-methyl-N,Ndimethyl-4-aminoazobenzene, was demonstrated (42). Aminoazo dye-bound alcohol dehydrogenase accounted for 45% of the total soluble protein-bound dye when this carcinogen was administered to rats. The binding to this protein was characterized as occurring specifically through alkylation of methionine 306. None of the other four methionine residues appeared to be alkylated. Thus, reaction between the dye and this enzyme occurs in a highly specific manner, just as in the several examples cited above. Not all electrophile-protein reactions involve carcinogens, of course. Affinity labeling, for example, has been widely used to identify reactive amino acids within drug or other ligand binding sites. These studies are not the subject of the present analysis because they take as their starting point two complementary reactants, the receptor protein and its specific substrate. The focus of this paper is reactions that occur by accident rather than by design, and within this narrower focus there are few, if any, other cases where a specific amino acid has been identified as the reactant center. One additional example, though, involving circumstantial evidence can be cited to further support the idea that even accidental adduct formation occurs with high specificity. Histones, in this example, are differentially alkylated by the enantiomers of BPDE (43). The (+) enantiomer reacts with both histones H2A and H3 in isolated nuclei prepared from hamster embryo cells grown in culture. Total binding to H3 is 32% of the binding to H2A. In contrast, the (-) enantiomer alkylates H2A almost exclusively. As in the reaction of BPDE with albumin, absolute configuration of the epoxide is clearly an important determinant of reactivity.

Conclusion The concept of precovalent binding is not a novel one, but it has only been invoked previously to explain specific experimental outcomes. The argument made here is that precovalent binding in the reaction of electrophiles with proteins is likely to be a common phenomenon, with exceptions arising primarily when a protein is devoid of a region into which the ligand can make an energetically favorable transfer from the aqueous surroundings. An

References (1) Berman, H. M., and Yang, P. R. (1981) The interaction of intercalating drugs with nucleic acids. Annu. Rev. Biophys. Bioeng. 10, 87-114. (2) Waring, M. J. (1981) DNA modification and cancer. Annu. Rev. Biochem. 50, 159-192. (3) Raney, K. D., Gopalakrishnan, S., Byrd, S., Stone, M. P., and Harris, T. M. (1990) Alteration of the aflatoxin cyclopentenone ring to a δ-lactone reduces intercalation with DNA and decreases formation of guanine N7 adducts by aflatoxin epoxides. Chem. Res. Toxicol. 3, 254-261. (4) Meehan, T., Gamper, H., and Becker, J. F. (1982) Characterization of reversible, physical binding of benzo[a]pyrene derivatives to DNA. J. Biol. Chem. 257, 10479-10485. (5) Geacintov, N. E. (1986) Is intercalation a critical factor in the covalent binding of mutagenic and tumorigenic polycyclic aromatic diol epoxides to DNA? Carcinogenesis 7, 759-766. (6) Iyer, R. S., Coles, B. F., Raney, K. D., Thier, R., Guengerich, F. P., and Harris, T. M. (1994) DNA adduction by the potent carcinogen aflatoxin B1: Mechanistic studies. J. Am. Chem. Soc. 116, 1603-1609. (7) Brown, J. R., and Shockley, P. (1982) Serum albumin: structure and characterization of its ligand binding sites. In Lipid-Protein Interactions (Jost, P. C., and Griffith, O. H., Eds.) pp 25-68, Wiley, New York. (8) Carter, D. C., and Ho, J. X. (1994) Structure of serum albumin. Adv. Protein Chem. 45, 153-215. (9) Perutz, M. F., Fermi, G., Abraham, D. J., Poyart, C., and Bursaux, E. (1986) Hemoglobin as a receptor of drugs and peptides: X-ray studies of the stereochemistry of binding. J. Am. Chem. Soc. 108, 1064-1078. (10) Smith, T. W., and Skubitz, K. M. (1975) Kinetics of interactions between antibodies and haptens. Biochemistry 14, 1496-1502. (11) Foote, J., and Milstein, C. (1991) Kinetic maturation of an immune response. Nature 352, 530-532. (12) Bradfield, C. A., Kende, A. S., and Poland, A. (1988) Kinetic and equilibrium studies of the Ah receptor-ligand binding: Use of [125I]2-iodo-7,8-dibromodibenzo-p-dioxin. Mol. Pharmacol. 34, 229237. (13) Scheider, W. (1977) Real-time measurement of dielectric relaxation of biomolecules: Kinetics of a protein-ligand binding reaction. Ann. N.Y. Acad. Sci. 303, 47-58. (14) Murthy, M. S. S., Calleman, C. J., Osterman-Golkar, S., Segerba¨ck, D., and Svensson, K. (1984) Relationship between ethylation of hemoglobin, ethylation of DNA and administered amount of ethyl methanesulfonate in the mouse. Mutat. Res. 127, 1-8. (15) Osterman-Golkar, S., Ehrenberg, L., Segerba¨ck, D., and Hallstrom, I. (1976) Evaluation of genetic risks of alkylating agents. II. Haemoglobin as a dose monitor. Mutat. Res. 34, 1-10. (16) Swain, C. G., and Scott, C. B. (1953) Quantitative correlation of relative rates. Comparison of hydroxide ion with other nucleophilic reagents toward alkyl halides, esters, epoxides and acyl halides. J. Am. Chem. Soc. 75, 141-147. (17) Roche, C. J., Zinger, D., and Geacintov, N. E. (1985) Enhancement of stability of 7β,8R-dihydroxy-9R,10R-epoxybenzo(a)pyrene by complex formation with serum albumin. Cancer Biochem. Biophys. 8, 35-40. (18) Eder, E., and Ku¨tt, W. (1989) The dependence of the mutagenicity of methanesulphonic acid esters in S. typhimurium TA100 on the alkylation mechanism. Chem.-Biol. Interact. 69, 45-59.

Perspective (19) McCleary, H. R., and Hammett, L. P. (1941) Rate and mechanism in the reactions of ethyl p-toluenesulfonate with water, hydroxyl ions and various halide ions. J. Am. Chem. Soc. 63, 2254-2262. (20) Ringe, D., Turesky, R. J., Skipper, P. L., and Tannenbaum, S. R. (1988) Structure of the single stable hemoglobin adduct formed by 4-aminobiphenyl in vivo. Chem. Res. Toxicol. 1, 22-24. (21) Day, B. W., Skipper, P. L., Rich, R. H., Naylor, S., and Tannenbaum, S. R. (1991) Conversion of a hemoglobin alpha-chain aspartate(47) ester to N-(2,3-dihydroxypropyl)asparagine as a method for the identification of the principal binding site for benzo[a]pyrene anti-diol epoxide. Chem. Res. Toxicol. 4, 359-363. (22) Hughes, G. J., De Jong, C., Fischer, R. W., Winterhalter, K. H., and Wilson, K. J. (1981) Modification by simetryn sulphoxide of a specific thiol group in rat haemoglobin. Biochem. J. 199, 6167. (23) Hutchins, D. A., Skipper, P. L., Naylor, S., and Tannenbaum, S. R. (1988) Isolation and characterization of the major fluoranthene-hemoglobin adducts formed in vivo in the rat. Cancer Res. 48, 4756-4761. (24) Hamboeck, H., Fischer, R. W., Di Iorio, E. E., and Winterhalter, K. H. (1981) The binding of s-triazine metabolites to rodent hemoglobins appears irrelevant to other species. Mol. Pharmacol. 20, 579-584. (25) Eyer, P. (1988) Detoxication of N-oxygenated arylamines in erythrocytes. An overview. Xenobiotica 18, 1327-1333. (26) Klehr, H., Eyer, P., and Scha¨fer, W. (1985) On the mechanism of reactions of nitrosoarenes with thiols. Formation of a common intermediate “semimercaptal”. Biol. Chem. Hoppe-Seyler 366, 755-760. (27) Eyer, P., and Ascherl, M. (1987) Reactions of para-substituted nitrosobenzenes with human hemoglobin. Biol. Chem. HoppeSeyler 368, 285-294. (28) Eyer, P. (1979) Reactions of nitrosobenzene with reduced glutathione. Chem.-Biol. Interact. 24, 227-239. (29) Manning, B. W., Lay, J. O., Dooley, K. L., and Kadlubar, F. F. (1988) The effect of glutathione on the binding of the nitroso metabolite of 4-aminobiphenyl to hemoglobin. The Toxicologist 8, 937. (30) Gibson, Q. H. (1960) The reactions of some aromatic C-nitroso compounds with haemoglobin. Biochem. J. 77, 519-526. (31) Skipper, P. L., Obiedzinski, M. W., Tannenbaum, S. R., Miller, D. W., Mitchum, R. K., and Kadlubar, F. F. (1985) Identification of the major serum albumin adduct formed by 4-aminobiphenyl in vivo in rats. Cancer Res. 45, 5122-5127. (32) Kadlubar, F. F., Casciano, D. A., Weis, C. C., Skipper, P. L., Tannenbaum, S. R., and Ketterer, B. (1985) Detoxification of carcinogenic aromatic amines by reaction with serum albumin. Proc. Am. Assoc. Cancer Res. 26, 424. (33) He, X. M., and Carter, D. C. (1992) Atomic structure and chemistry of human serum albumin. Nature 358, 209-215. (34) Day, B. W., Skipper, P. L., Zaia, J., Singh, K., and Tannenbaum, S. R. (1994) Enantiospecificity of covalent adduct formation by

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(36)

(37)

(38)

(39)

(40)

(41)

(42)

(43)

(44)

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benzo[a]pyrene anti-diol epoxide with human serum albumin. Chem. Res. Toxicol. 7, 829-835. Zaia, J., and Biemann, K. (1994) Proteinase K activity inhibited near amino acids carrying large substituents: Three PAH diolepoxides covalently modify His-146 human serum albumin residue. J. Am. Chem. Soc. 116, 7407-7408. Zaia, J., and Biemann, K. (1994) Characteristics of high energy collision-induced dissociation tandem mass spectra of polycyclic aromatic hydrocarbon diolepoxide adducted peptides. J. Am. Soc. Mass Spectrom. 5, 649-654. Zaia, J., and Biemann, K. (1994) Towards the mass spectrometric identification of human serum albumin covalent binding sites for biologically relevant levels of polycyclic aromatic hydrocarbon diolepoxides. Proc. 42nd ASMS Conf. Mass Spectrom. Allied Top., 634. Ding, A., Ojingwa, J. C., McDonagh, A. F., Burlingame, A. L., and Benet, L. Z. (1993) Evidence for covalent binding of acyl glucuronides to serum albumin via an imine mechanism as revealed by tandem mass spectrometry. Proc. Natl. Acad. Sci. U.S.A. 90, 3797-3801. Miller, E. C., and Miller, J. A. (1947) The presence and significance of bound aminoazo dyes in the livers of rats fed pdimethylaminoazobenzene. Cancer Res. 7, 468-480. Scribner, J. D., Miller, J. A., and Miller, E. C. (1965) 3-Methylmercapto-N-methyl-4-aminoazobenzene: an alkaline-degradation product of a labile protein-bound dye in the livers of rats fed N,Ndimethyl-4-aminoazobenzene. Biochem. Biophys. Res. Commun. 20, 560-565. Lin, J.-K., Miller, J. A., and Miller, E. C. (1968) Studies on structures of polar dyes derived from the liver proteins of rats fed N-methyl-4-aminoazobenzene. II. Identity of synthetic 3-(homocystein-S-yl)-N-methyl-4-aminoazobenzene with the major polar dye P2b. Biochemistry 7, 1889-1895. Coles, B., Beale, D., Miller, D., Lay, J., Kadlubar, F., Aitken, A., and Ketterer, B. (1987) The binding of an aminoazo dye carcinogen to a specific methionine residue in rat liver alcohol dehydrogenase in vivo. Chem.-Biol. Interact. 64, 181-192. Kootstra, A., MacLeod, M. C., Iyer, R., Selkirk, J. K., and Slaga, T. J. (1982) Selective modification of nuclear proteins by polycyclic aromatic hydrocarbons and by benzo[a]pyrene diol epoxides. Carcinogenesis 3, 821-824. Richmond, T. J., Finch, J. T., Rushton, B., Rhodes, D., and Klug, A. (1984) Structure of the nucleosome core particle at 7 Å resolution. Nature 311, 532-537. Arents, G., Burlingame, R. F., Wang, B.-C., Love, W. E., and Moudrianakis, E. N. (1991) The nucleosomal core histone octamer at 3.1 Å resolution: A tripartite protein assembly and a lefthanded superhelix. Proc. Natl. Acad. Sci. U.S.A. 88, 10148-10152.

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