Biosynthesis of porphyrins and related macrocycles. Part 53. Stereochemical studies on the enzymic formation of hydroxymethylbilane, the precursor of uroporphyrinogen III

Abstract

A new synthesis of porphobilinogen 1 (PBG) is described that allows the preparation of (11R)-[11-³H₁]PBG 1a and its (11S )-enantiomer 1b. Their enantiomeric purities are determined by degradation of their immediate synthetic precursors by way of ³H-labelled glycines to yield two samples of ³H-labelled glycolic acid 16. The enzyme glycolate oxidase, known to remove H_R stereospecifically from the methylene group of glycolic acid in forming glyoxylic acid 17, is then used to assay the configurations of these two samples. Each ³H-labelled PBG 1a and 1b is converted by hydroxymethylbilane synthase into hydroxymethylbilane 5a and 5b. Methods are devised for the isolation of this labile product from water and for its subsequent degradation to two further samples of glycolic acid. These are assayed enzymically to prove that there is overall retention of configuration as the aminomethyl carbon of PBG 1 enzymically affords the hydroxymethyl centre of the bilane 5. Thus, the two covalent bonds that are formed in this whole process must both involve reactions with retention of configuration or both with inversion. The significance of these results is discussed.

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