Purified 5-aminolaevulinate dehydratase (porphobilinogen synthase, EC 184.108.40.206) from human erythrocytes was incubated initially with limiting amounts of 5-amino [5-14C]laevulinate in a rapid-mixing apparatus. The single-turnover reaction with respect to the bound labelled 5-aminolaevulinate was completed by the addition of unlabelled 5-aminolaevulinate and the resulting radioactive porphobilinogen was isolated and degraded. The 14C label was found to be located predominantly at C-2 of the product, demonstrating that, of the two substrate molecules participating in the reaction, the 5-aminolaevulinate molecule initially bound to the enzyme provides the propionic acid ‘side’ of the porphobilinogen. The same enzyme-[14C]substrate species that yields regiospecific porphobilinogen may be trapped by reaction with NaBH4, showing that the substrate molecule initially bound to the enzyme does so in the form of a Schiff base. A conventional incubation with 5-amino[5-14C]laevulinate yielded porphobilinogen with an equal distribution of the label between C-2 and C-11. The reaction mechanism of the human erythrocyte 5-aminolaevulinate dehydratase thus follows the same course as that of other dehydratases studied in our laboratory by using single-turnover techniques.
Research Article| May 01 1985
Mechanism of action of 5-aminolaevulinate dehydratase from human erythrocytes
Biochem J (1985) 227 (3): 1015–1020.
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P M Jordan, P N Gibbs; Mechanism of action of 5-aminolaevulinate dehydratase from human erythrocytes. Biochem J 1 May 1985; 227 (3): 1015–1020. doi: https://doi.org/10.1042/bj2271015
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