A radiochemical method for the measurement of coproporphyrinogen oxidase and the utilization of substrates other than coproporphyrinogen III by the enzyme from rat liver

[14C2] Coproporphyrin 111, 14C-labelled in the carboxyl carbon atoms of the 2- and 4-propionate substituents, was prepared by stepwise modification of the vinyl groups of protoporphyrin IX. The corresponding porphyrinogen was used as substrate in a specific sensitive assay for coproporphyrinogen oxidase (EC 1.3.3.3) in which the rate of production of 14CO2 is measured. With this method, the Ksub(m) of the enzyme from rat liver for coproporphyrinogen III is 1.2μM. Coproporphyrin III is a competitive inhibitor of the enzyme (Ksub(i) 7.6μM)> Apparent Ksub(m) values for other substrates were measured by a mixed-substrate method: that for coproporphyrinogen IV is 0.9μM and that for harderoporphyrinogen 1.6μM. Rat liver mitochondria convert pentacarboxylate porphyrinogen III into dehydroosicoproporphyrinogen at a rate similar to that for the formation of protoporphyrinogen IX from coproporphyrinogen III. Mixed-substrate experiments indicate that this reaction is catalysed by coproporphyrinogen oxidase and that the Ksub(m) for this substrate is 29μM. It is suggested that the ratio of the concentration of pentarcarboxylate porphyrinogen III to coproporphyrinogen III in the hepatocyte determines the relative rates of formation of dehydroisocoproporphyrinogen and protoporphyrinogen IX. (author)