Normal and abnormal heme biosynthesis, 1. Synthesis and metabolism of di- and monocarboxylic porphyrinogens related to coproporphyrinogen-III and harderoporphyrinogen: A model for the active site of coproporphyrinogen oxidase

Coproporphyrinogen oxidase (copro'gen oxidase), which catalyses the convers ion of coproporphyrinogen-III via a monovinylic intermediate to protoporphy rinogen-IX, is one of the least well understood enzymes in the heme biosynt hetic pathway. To develop a model for the substrate recognition and binding recognition for this enzyme, a series of substrate analogues were prepared with two alkyl substituents on positions 13 and 17 in place of the usual p ropionate residues. Although the required substrate probes are porphyrinoge ns (hexahydroporphyrins), the corresponding porphyrin methyl esters were in itialy synthesized via a,c-biladiene intermediates. These were hydrolyzed a nd reduced with 3% sodium amalgam to give the unstable porphyrinogens neede d for the biochemical investigations. These modified structures were metabo lized by avian preparations of copro'gen oxidase to give monovinylic produc ts, but the second propionate residue was not further metabolized. In three cases, the metabolites were isolated and further characterized by proton N MR spectroscopy and mass spectrometry. When methyl or ethyl groups were pla ced at the 13 and 17 positions, the resulting porphyrinogens were very good substrates (although the ethyl version, mesoporphyrinogen-VI, gave slightl y better results), but when propyl units were introduced metabolism was sig nificantly inhibited and the butyl-substituted structure was only slightly transformed after long incubation periods. These results suggest the presen ce of an active-site lipophobic region near the catalytic site for copro'ge n oxidase. The observation that the related 3-vinyl- and 3-ethylporphyrinog ens with 13,17-diethyl substituents were not substrates for this enzyme con firmed the need for a second propionate residue to hold the substrate in pl ace at the catalytic site.