Studies on the biosynthesis of uroporphyrinogen III

Berry, Alan (1983) Studies on the biosynthesis of uroporphyrinogen III. University of Southampton, Doctoral Thesis.

Abstract

Porphobilinogen deaminase, a key enzyme in porphyrin biosynthesis, was purified from Rhodopseudomonas spheroides, and was used to generate preuroporphyrinogen, the recently discovered intermediate in tetrapyrrole biosynthesis. The optimum conditions for the conversion of porphobilinogen into preuroporphyrinogen were determined. Preuroporphyrinogen was then shown to act as the substrate for the uroporphyrinogen III cosynthetases from a variety of organisms forming uroporphyrinogen III in good yield and thus establishing preuroporphyrinogen as a universal intermediate in the porphyrin biosynthetic pathway in all living systems. Uroporphyrinogen III cosynthetase was semi-purified from human erythrocytes, and a kinetic study of uroporphyrinogen III formation from preuroporphyrinogen was made. The Km of cosynthetase for preuroporphyrinogen was determined. A comparison of the levels of uroporphyrinogen III cosynthetase in erythrocytes from normal human subjects and from a congenital erythropoietic porphyriac individual showed a decreased level of cosynthetase in this porphyria. The nature of the association between the enzyme porphobilinogen deaminase and its substrate, porphobilinogen, was investigated and the presence of three catalytically viable covalent enzyme-intermediate complexes was demonstrated. In order to fully characterise these complexes, [3,5-14 C2] porphobilinogen was biosynthesised from 5-amino [4- 4C] levulinic acid using purified bovine 5-amino levulinic acid dehydratase, and the labelled porphobilinogen was used to generate [14 C] labelled deaminase-intermediate complexes. These were isolated individually by polyacrylamide gel electrophoresis and each was converted into protoporphyrin IX using excess unlabelled porphobilinogen and a cell free system from R. spheroides. Protoporphyrin IX was isolated and chemically degraded in order to determine which of thefour pyrrole rings was [14C] labelled. The results proved unambiguously that the complexes were composed of porphobilinogen deaminase covalently linked to either one, two or three pyrrole rings.The ability to separate these enzyme-intermediate complexes alloweda mechanistic study using [2-3H] and [2-ZH] porphobilinogen to be made. The experiments showed that as ring 'B' condenses with ring 'A', the tritium at C-2 on ring 'A' is lost. Similarly, as ring 'C' condenses with rings 'A' and 'B', and ring 'D' with rings 'A', 'B' and 'C', the tritium at the free a-position of the growing tetrapyrrole is lost at each step, resulting in preuroporphyrinogen with a tritium at the freea-position. It was also shown that this tritium is lost on conversion of preuroporphyrinogen either into uroporphyrinogen I chemically or into uroporphyrinogen III by uroporphyrinogen III cosynthetase.

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