Factors determining the sequence of oxidative decarboxylation of the 2- and 4-propionate substituents of coproporphyrinogen III by coproporphyrinogen oxidase in rat liver

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Abstract
Coproporphyrinogen oxidase (EC 1.3.3.3) catalyzes the oxidative decarboxylation of the 2- and 4-propionate substituents of coproporphyrinogen III to form protoporphyrinogen IX. A 4-propionate-substituted porphyrinogen, harderoporphyrinogen, which is also a substrate for coproporphyrinogen oxidase, is formed during the reaction. Synthetic [14C]coproporphyrinogens III, specifically labelled in the carboxyl carbon atoms of the 2- or 4-propionate substituents, were used to measure rate of decarboxylation of each substituent by rat liver coproporphyrinogen oxidase. Experimental results, together with the recognition that in all known substrates of coproporphyrinogen oxidase only those propionate groups flanked by a specific arrangement of substituents are decarboxylated, indicate that the 4-propionate group of coproporphyrinogen III cannot be attacked until the 2-propionate group was decarboxylated. Production of 14CO2 from the substrate labelled in the 2-propionate group measures the formation of harderoporphyrinogen, whereas 14CO2 from the 4-propionate-labelled substrate measures protoporphyrinogen IX formation. Rate of harderoporphyrinogen formation is about twice that of protoporphyrinogen, and this ratio is unchanged by varying the concentration of coproporphyrinogen III or by competitive inhibition of the enzyme. When coproporphyrinogen III is present in an excess, 2 fractions of harderoporphyrinogen can be distinguished. One accumulates during the reaction, and the other, which is destined to become protoporphyrinogen IX, does not equilibrate with added harderoporphyrinogen. Both decarboxylations may take place at the same active center, which becomes temporarily inaccessible to coproporphyrinogen III and added harderoporphyrinogen, and the molecule apparently rotates after the 1st decarboxylation to allow the 2nd to take place.