Biosynthesis of porphyries and related macrocycles. Part 18. Proof by spectroscopy and synthesis that unrearranged hydroxymethylbilane is the product from deaminase and the substrate for cosynthetase in the biosynthesis of uroporphyrinogen-III

Abstract

When the enzyme deaminase acts alone on porphobilinogen, it releases a transient intermediate into the medium which is unaffected by further treatment with a large excess of deaminase. The intermediate undergoes rapid ringclosure chemically (t_½ca. 4 min) to form uroporphyrinogen-I. ¹³C Spectroscopic studies on the intermediate generated from ¹³C labelled porphobilinogen combined with synthesis of labelled standards for determination of chemical shifts establish its structure to be a linear tetrapyrrole, the unrearranged hydroxymethylbilane. Other workers deduced a different, cyclic structure (preuro'gen) which is shown here to be incorrect by chemical studies, ¹³C spectroscopy and ¹³C:¹⁵N double-labelling experiments. That the intermediate is the unrearranged hydroxymethylbilane is confirmed by its unambiguous synthesis. The natural and synthetic samples of this bilane are shown to be excellent and identical substrates for cosynthetase (free from deaminase) with production of uroporphyrinogen-III. Thus, deaminase is the enzyme for assembly of four porphobilinogen units to the linear tetrapyrrole stage and cosynthetase is the ring-closing and rearranging enzyme. Two proposals are discussed for the mechanism of inversion of the terminal ring-D of the hydroxymethylbilane in the formation of uroporphyrinogen-III.