The use of stereospecifically labelled precursors

ALA synthase is a pyridoxal phosphate-dependent enzyme and promotes Schiff-base formation between its coenzyme and glycine (67 in Fig. 37). Nucleophilicity at C-2 of the glycine could be generated either by decarboxylation or by abstraction of a proton. In the first case 5-aminolaevulinic acid would retain both methylene protons of glycine, in the second, one of the protons would be lost to the medium (Fig. 37). Acylation of the pyridoxal-bound intermediate (68 or 69) by succinyl-CoA would constitute the next step and this could be followed either by direct hydrolysis of the Schiff-base or by decarboxylation with subsequent hydrolysis depending on which course was chosen in the first stage of the reaction. 

To elucidate the mechanistic details (R)- and (S)-[2-3H]glydnes were prepared [79] (see also chapter by Floss and Vederas) and reacted with succinyl-CoA on ALA synthase. It was shown that tritium was retained in ALA when (S)-[2-3H]glydne was used as substrate but it was lost when (R )-[2-3H]glycine was the starting material. 

A further question concerns the fate of the tritium in the second molecule of (5S)-[5-3H]ALA during the dehydratase reaction. The C-5 atom of the ALA molecules becomes the C-2 atom of PBG and loses one of its enantiotopic protons, while the ring is aromatized. When (5S)-[5-3H]ALA was reacted on the dehydratase and the product was isolated by careful chromatography on cellulose, the resulting PBG retained the tritium also at position 2 [81]. This means that the aromatization of the presumptive intermediate 71 takes place in an enzyme-bound state and involves specific abstraction of the 2-HRe atom (Fig. 39). 

Though the pertinent experiment in the corrin series has not yet been reported, a record of the analogous experiment in the porphyrin series has appeared [84], In contrast to corrins, in haems all four acetate side chains of the parent urogen III have been decarboxylated to methyl groups. In principle, these could be carved out as acetic acid and analysed for chirality if the starting succinate had been stereospecifically labelled with deuterium as well as with tritium. The synthesis of (2/ )-[2-2H,2-3 H]succinic acid was achieved by incubating (37 ,S)-[3-3 H]- 

Barnard and Akhtar [84] speculate that the same group (X) of the enzyme is involved in the deprotonation of the carboxyl group and in the stereospecific protonation of the vinylogous enamine (75) (Fig. 41). 

---