Stereochemical and mechanistic studies on the decarboxylation of uroporphyrinogen III in haem biosynthesis

Abstract

The substrate stereochemistry of porphyrinogen carboxy-lyase which catalyses the decarboxylation of the four acetate side chains of uroporphyrinogen III was determined. (2R)-[2-³H₁,2-²H₁]Succinic acid was prepared from (3RS)-[³H₁]-2-oxoglutarate using the stereospecific exchange reaction catalysed by NADP⁺-dependent isocitrate dehydrogenase, followed by decarboxylation. The (2R)-[2-³H₁,2-²H₁]succinic acid was incorporated into haem using a cell-free preparation obtained from anaemic chicken erythrocytes. The biosynthetic haem was degraded initially to haematinic acid and ethylmethyl maleimide. A method involving ozonolysis was developed for the further degradation of these imide rings, with minimal concomitant proton labilisation, to give acetic acid. The chirality of the asymmetric methyls in acetic acid samples was determined using malate synthetase and fumarase, which catalyse reactions of known stereochemical course. The chiral analysis established that in the porphyrinogen carboxy-lyase reaction both protons at each methylene of uroporphyrinogen III remain intact in coproporphyrinogen III and that the reaction proceeds with retention of configuration at the methylene carbons of all four acetate side chains. In the light of these results a possible mechanism for the porphyrinogen carboxy-lyase reaction is proposed.