Hydroxamic acid production by α-ketoglutarate dehydrogenase. Part 2. Evidence for an electrophilic reaction intermediate at the enzyme active site

Abstract

The α-ketoglutarate dehydrogenase-catalyzed conversion of 4-chloronitrosobenzene (1) into the hydroxamic acid (3) and the Bamberger-rearrangement product (6) was investigated by use of radio-tracer methods and nucleophilic trapping agents. ¹⁴C-Labelled 4-chloronitrosobenzene (1) failed to give any significant incorporation of radiolabel into the protein of the enzyme, or into calf thymus DNA. The production of a third and highly polar metabolite during this reaction was confirmed; however, the structure of this metabolite has not been elucidated. In the presence of high concentrations of halide salts, the product distribution for the enzymic reaction was markedly altered. In the order I^– > Br^– > Cl^–, halides inhibited the production of the rearrangement product (6) and of the unknown polar product. The inhibition of the formation of these products was accompanied by a considerable increase in the amount of hydroxamic acid (3), and by the production of a new metabolite, the structure of which was dependent upon the halide employed in the reaction. In the case of both Br^– and Cl^–, the new metabolite [(8a) and (8b), respectively] was indicative of the trapping of an enzyme-generated electrophile by halide anion. In the case of I^–, the initial trapping of the electrophilic species was followed by a redox process to give 4-chloroaniline (9).