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

Abstract

[14C2]Coproporphyrin III, 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 Km of the enzyme from rat liver for coproporphyrinogen III is 1.2 micron. Coproporphyrin III is a competitive inhibitor of the enzyme (Ki 7.6 micron). Apparent Km values for other substrates were measured by a mixed-substrate method: that for coproporphyrinogen IV is 0.9 micron and that for harderoporphyrinogen 1.6 micron. Rat liver mitochondria convert pentacarboxylate porphyrinogen III into dehydroisocoproporphyrinogen 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 Km for this substrate is 29 micron. It is suggested that the ratio of the concentration of pentacarboxylate porphyrinogen III to coproporphyrinogen III in the hepatocyte determines the relative rates of formation of dehydroisocoproporphyrinogen and protoporphyrinogen IX.

Crystal Structure of the Oxygen-dependant Coproporphyrinogen Oxidase (Hem13p) of Saccharomyces cerevisiae

“…If the reaction intermediate is able to reposition within the active-site cavity, this architecture would explain why the first decarboxylation, on the pyrrole A ring, is rate-limiting and rapidly followed by decarboxylation of the pyrrole B ring propionate (13,14). One potential advantage of the substrateinduced conformational change is that it might generate a specific pathway and binding site for the molecular oxygen cofactor, such as seen for cholesterol oxidase (42,43).…”

“…Form I and II monomers overlap with a r.m.s.d. of ϳ1.5 Å on all pairs of C␣ atoms except those before Arg 13 and between Gln 77 and Lys 110 , inclusive. The 12 N-terminal residues project in very different directions in the two crystal forms, apparently because of different lattice constraints.…”

“…There are reports of requirements for copper (8) and manganese (9), although other studies found no metal ion or other cofactor dependence (except O 2 ) for odCPO activity (10 -12). Regardless of mechanistic details, the first decarboxylation has been shown to be the rate-limiting step for the overall reaction, and transient formation of the 3-carboxyl intermediate harderoporphyrinogen has been demonstrated (13,14).…”

Computational Characterization of the Substrate-Binding Mode in Coproporphyrinogen III Oxidase

“…The solvent-exposure of the ring B methyl group in this binding mode is not consistent with the experimentally verified requirement of a small non-polar substituent (rather than e.g. acetate 13 ) at that position. The non-involvement of H131, R135, R275 and D274 in this substrate binding mode also argues against its mechanistical relevance.…”

“…-amino acids numbered according to the mature yeast protein) are critically important for substrate binding and/or catalysis, and extensive investigations with synthetic pyrroles 5,[13][14][15][16] have further shown that the active site contains different regions important for substrate recognition: the "Z" region can accommodate small nonpolar groups (R=H, methyl, ethyl vinyl) and discriminates against charged groups (like acetate and propionate), a neighbouring "Y" region binds the propionate group undergoing oxidation, and a third "X" region requires the presence of a propionate (or vinyl 16 ) group (Scheme 2). Due to these constraints, only tetrapyrroles bearing a R-Me-Pr-Me-Pr/Vin sequence of substituents (where R is a small nonpolar group) may be metabolized by the enzyme.…”