Biosynthesis of porphyrins and related macrocycles. Part 25. Synthesis of analogues of coproporphyrinogen-III and studies of their interaction with coproporphyrinogen-III oxidase from Euglena gracilis
“…Other substrates have been reported (Table ), − and these suggest that a specific sequence of peripheral substituents are necessary for enzyme−substrate recognition. Initially, the sequence R Me-P Me (where R can be H, Me, vinyl (V) or Et but not a bulky propionate (P) side chain) was proposed as a minimal requirement for metabolism, but the necessity of a second propionate residue was subsequently recognized. 13b,, For the known substrates listed in Table , the necessary sequence R Me-P Me-P has been highlighted in bold; the selected nonsubstrates all interupt this sequence in one way or another. 1 Type Isomers of Coproporphyrinogen That Are Not Metabolized by Copro'gen Oxidases 4 Metabolism of Coproporphyrinogen-IV…”
Section: Substrate Recognition: a Model For The Active
Site Of Copro...mentioning
confidence: 98%
“…In contrast, while copro'gen-IV appears to be an equally good substrate for this enzyme and is eventually converted into proto'gen-XIII, the type IV “hardero'gen” accumulates to approximately 40% of total porphyrinogen content part way through the metabolism . Other substrates have been reported (Table ), − and these suggest that a specific sequence of peripheral substituents are necessary for enzyme−substrate recognition. Initially, the sequence R Me-P Me (where R can be H, Me, vinyl (V) or Et but not a bulky propionate (P) side chain) was proposed as a minimal requirement for metabolism, but the necessity of a second propionate residue was subsequently recognized. 13b,, For the known substrates listed in Table , the necessary sequence R Me-P Me-P has been highlighted in bold; the selected nonsubstrates all interupt this sequence in one way or another.…”
Section: Substrate Recognition: a Model For The Active
Site Of Copro...mentioning
Coproporphyrinogen oxidase (copro'gen oxidase), which catalyses the conversion of coproporphyrinogen-III via a monovinylic intermediate to protoporphyrinogen-IX, is one of the least well
understood enzymes in the heme biosynthetic 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 propionate residues.
Although the required substrate probes are porphyrinogens (hexahydroporphyrins), the corresponding porphyrin methyl esters were initialy synthesized via a,c-biladiene intermediates. These
were hydrolyzed and reduced with 3% sodium amalgam to give the unstable porphyrinogens needed
for the biochemical investigations. These modified structures were metabolized by avian preparations of copro'gen oxidase to give monovinylic products, but the second propionate residue was not
further metabolized. In three cases, the metabolites were isolated and further characterized by
proton NMR spectroscopy and mass spectrometry. When methyl or ethyl groups were placed at
the 13 and 17 positions, the resulting porphyrinogens were very good substrates (although the
ethyl version, mesoporphyrinogen-VI, gave slightly better results), but when propyl units were
introduced metabolism was significantly inhibited and the butyl-substituted structure was only
slightly transformed after long incubation periods. These results suggest the presence of an active-site lipophobic region near the catalytic site for copro'gen oxidase. The observation that the related
3-vinyl- and 3-ethylporphyrinogens with 13,17-diethyl substituents were not substrates for this
enzyme confirmed the need for a second propionate residue to hold the substrate in place at the
catalytic site.
“…Other substrates have been reported (Table ), − and these suggest that a specific sequence of peripheral substituents are necessary for enzyme−substrate recognition. Initially, the sequence R Me-P Me (where R can be H, Me, vinyl (V) or Et but not a bulky propionate (P) side chain) was proposed as a minimal requirement for metabolism, but the necessity of a second propionate residue was subsequently recognized. 13b,, For the known substrates listed in Table , the necessary sequence R Me-P Me-P has been highlighted in bold; the selected nonsubstrates all interupt this sequence in one way or another. 1 Type Isomers of Coproporphyrinogen That Are Not Metabolized by Copro'gen Oxidases 4 Metabolism of Coproporphyrinogen-IV…”
Section: Substrate Recognition: a Model For The Active
Site Of Copro...mentioning
confidence: 98%
“…In contrast, while copro'gen-IV appears to be an equally good substrate for this enzyme and is eventually converted into proto'gen-XIII, the type IV “hardero'gen” accumulates to approximately 40% of total porphyrinogen content part way through the metabolism . Other substrates have been reported (Table ), − and these suggest that a specific sequence of peripheral substituents are necessary for enzyme−substrate recognition. Initially, the sequence R Me-P Me (where R can be H, Me, vinyl (V) or Et but not a bulky propionate (P) side chain) was proposed as a minimal requirement for metabolism, but the necessity of a second propionate residue was subsequently recognized. 13b,, For the known substrates listed in Table , the necessary sequence R Me-P Me-P has been highlighted in bold; the selected nonsubstrates all interupt this sequence in one way or another.…”
Section: Substrate Recognition: a Model For The Active
Site Of Copro...mentioning
Coproporphyrinogen oxidase (copro'gen oxidase), which catalyses the conversion of coproporphyrinogen-III via a monovinylic intermediate to protoporphyrinogen-IX, is one of the least well
understood enzymes in the heme biosynthetic 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 propionate residues.
Although the required substrate probes are porphyrinogens (hexahydroporphyrins), the corresponding porphyrin methyl esters were initialy synthesized via a,c-biladiene intermediates. These
were hydrolyzed and reduced with 3% sodium amalgam to give the unstable porphyrinogens needed
for the biochemical investigations. These modified structures were metabolized by avian preparations of copro'gen oxidase to give monovinylic products, but the second propionate residue was not
further metabolized. In three cases, the metabolites were isolated and further characterized by
proton NMR spectroscopy and mass spectrometry. When methyl or ethyl groups were placed at
the 13 and 17 positions, the resulting porphyrinogens were very good substrates (although the
ethyl version, mesoporphyrinogen-VI, gave slightly better results), but when propyl units were
introduced metabolism was significantly inhibited and the butyl-substituted structure was only
slightly transformed after long incubation periods. These results suggest the presence of an active-site lipophobic region near the catalytic site for copro'gen oxidase. The observation that the related
3-vinyl- and 3-ethylporphyrinogens with 13,17-diethyl substituents were not substrates for this
enzyme confirmed the need for a second propionate residue to hold the substrate in place at the
catalytic site.
“…However, once in every 26 turnovers, on average, the cytochrome is inactivated, by alkylation of the haem moiety. The porphyrin that was obtained by demetallation and esterification of the inactivated cytochrome has been shown" to be protoporphyrin dimethyl ester in which there is an acylmethyl group exclusively on ring A, as in (37) [except for the reaction with acetylene itself (35; R = H), which also alkylates other nitrogen atoms to a small extent]. Terminal alkenes are epoxidized by cytochrome P-450, and again alkylation of the haem occasionally occurs to give, in this case, a 2-hydroxyalkyl derivative.…”
“…However, this model does not account for the influence of alkyl groups at positions 13 and 17 on the first oxidative decarboxylation, which is favored when the substituents are Et or Me (1a and 1b), but less so for the dipropyl substrate 1c and greatly inhibited for dibutyl porphyrinogen 1d. The 8-butyrate derivative 4a of copro'gen-III has been shown to be a substrate for CPO preparations from Euglena gracilis, affording 3-vinylporphyrinogen 5a, 34 However, the branched substituents in porphyrinogens 4b and 4c did allow these species to be metabolized to a limited extent to give primarily the monovinyl products 5b and 5c, respectively (Scheme 4). 35 These data show that extended or branched carboxylate units can be accommodated at site X in the binding model, 12,35 but the modified porphyrinogens are far less effective substrates for CPO.…”
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