Nucleophilic addition, covalent binding

Thorn KA, PJ Pettigrew, WS Goldenberg, EJ Weber (1996) Covalent binding of aniline to humic substances. 2. N NMR studies of nucleophilic addition reactions. Environ Sci Technol 30 1764-1775. 

When reactive metabolites are formed by metabolic activation, some of them can escape from the active site and bind to external protein residues or be trapped by reduced glutathione (GSH) or other nucleophiles. The remaining molecules that are not released from the active site will cause the suicide inhibition [7]. The ratio of the number of reactive molecules remaining in the active site and those escaping is a measure of the reactivity of the intermediates formed. The addition of scavengers or GSH to the incubation mixture does not affect and cannot prevent the CYP mechanism-based inhibition. However, GSH can reduce the extent of the nonspecific covalent binding to proteins by those reactive molecules that escape from the active site. In contrast, addition of substrates or inhibitors that compete for the same catalytic center usually results in reduction of the extent of inhibition. 

Thorn, K. A., Goldenberg, W. S., Younger, S. J., and Weber, E. J. (1996a). Covalent binding of aniline to humic substances—Comparison of nucleophilic addition, enzyme-, and metal-catalyzed reactions by N-15 NMR. ACS Symp. Ser. 651, 299-326. 

In addition to DNA adducts that occur as a result of covalent binding of reactive intermediates generated by oxidation or conjugation of parent compounds to DNA, reactive oxygen species produced during xenobiotic metabolism can also react with nucleophilic biomolecules. 

Substrate binding and activation are followed by attack of the carboxylate side chain of Asp-145 at the benzoyl C-4 atom to give an enzyme-stabilized Meisenheimer intermediate (EMc) (Figure 8). Indeed, a site-directed mutant in which Asp-145 has been replaced by an alanine is catalytically inactive." Ketonization of the EMc results in rearomatization of the benzoyl ring and expulsion of the chloride. This nucleophilic addition-elimination mechanism (SNAr-type reaction) results in a second covalent (aryl-enzyme) intermediate, which is subsequently hydrolyzed by a water molecule that is activated by His-90 to give the free enzyme and the product. The existence of a covalent aryl-enzyme intermediate has been inferred from 0-labeling studies (similar to those described for haloalkane and haloalcohol dehalogenase) and from the direct measurement of the aryl-enzyme... 

With this approach we and others have discovered that several commonly used drugs can cause tissue damage through the formation of metabolites (Table 1). In addition the studies on the effects of inducers, inhibitors and potential nucleophiles on the covalent binding of chemically reactive metabolites formed vitro have helped us to understand the characteristics and properties of the chemically reactive metabolites and the enzymes that catalyze their formation and inactivation. 

Evidence is increasing to support the formation of arene oxides during the metabolism of certain PCBs (14). In addition, chemical synthesis of certain PCB arene oxides has been completed and these oxides show the expected chemical properties consistent with their potential for rearrangement and covalent binding to various nucleophiles. 

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