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 (4 ca. 4 min) to form uraporphyrinogen-I. '3C 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, l3C 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-Ill. 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- Ill.THE formation of uroporphyrinogen-111 (uro'gen-111) (3) and ammonia from four molecules of porphobilinogen (PBG) (1) is catalysed in living systems by two proteins, deaminase and cosynthetase. Cosynthetase is easily destroyed by heat-treatment and deaminase alone acts on PBG (1) to produce uroporphyrinogen-I (uro'gen-I) * This proposal grew as a result of a valuable discussion with Dr. D. C. Williams (Trinity College, Dublin) who we warmly thank, on the possible involvement of methylene-tetrahydrofolate in porphyrin biosynthesis.

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The labile unrearranged hydroxymethylbilane (5) is synthesised unambiguously, is proved to be identical with the product from deaminase acting on porphobilinogen, and is shown to be the substrate for deaminase-free cosynthetase which quantitatively ringcloses (5) with rearrangement to uro'gen-I11 (7).EARLIER work showed that uro'gen-I11 (7), the precursor of the natural porphyrins, chlorins, and vitamin B,,,l is biosynthesised by head-to-tail assembly of 4 porphobilinogen units, P B G (1) , starting at ring-ti and building round to ring-D2 to produce a bilane (4) followed by intramolecular rearrangement to reverse ring-D . 3 Two enzymes, deaminase and cosynthetase, work together to produce this result.4 Recently it was found5 that deaminase

Six isomeric aminomethylbilanes have been built by unambiguous synthesis. One bilane has the unrearranged structure corresponding to straightforward head-to-tail assembly of four units of porphobilinogen ; the other five bilanes have one or more of the pyrrole rings reversed relative to the unrearranged bilane. The action of deaminasecosynthetase on these bilanes has been studied and the results showed that (a) the enzyme system ring closes the unrearranged bilane far more efficiently than it closes any of the five isomeric bilanes, (b) under the best conditions the product formed enzymically from the unrearranged bilane is almost pure uroporphyrinogen-Ill, and (c) a transient intermediate is formed enzymically from the unrearranged bilane which can be detected kinetically. Further support is thereby given to the earlier conclusion that the intramolecular rearrangement which produces the natural type-Ill porphyrins occurs at the unrearranged tetrapyrrole level.The enzymic results for the other bilanes are also described together with an improved method for analytical separation of the four isomeric esters of coproporphyrin. t Similar preparations of deaminase-cosynthetase were also used in Part 16 and Part 15 1 4 of this Series. * This solution was freshly prepared from sodium metabisulphite; 0.1 ml contained just sufficient reagent t o decolourise 0.4 ml of the iodine solution.* The non-systematic name ' tripyrrene ' has been retained in order to ensure consistency with earlier parts of the Series. Such compounds should be named according to the IUPAC rules as ' trip yrrins . '

Wenn Desaminase auf Porphobilinogen (I) einwirkt, wird intermediär eine Zwischen‐ Verbindung freigesetzt, die nach 13C‐NMR‐spektroskopischen und chemischen (Synthese von markierten Vorstufen) Untersuchungen die Struktur des linearen Tetrapyrrols (IVb), eines (im Ring Dnicht umgelagerten) Hydroxymethylbilans, hat.