Jordan P M, Gibbs P N
Biochem J. 1985 May 1;227(3):1015-20. doi: 10.1042/bj2271015.
Purified 5-aminolaevulinate dehydratase (porphobilinogen synthase, EC 4.2.1.24) 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.
将从人红细胞中纯化得到的5-氨基酮戊酸脱水酶（胆色素原合酶，EC 4.2.1.24），最初在快速混合装置中与限量的5-氨基[5-¹⁴C]酮戊酸一起孵育。通过加入未标记的5-氨基酮戊酸，完成了与结合的标记5-氨基酮戊酸相关的单周转反应，并分离并降解了生成的放射性胆色素原。发现¹⁴C标记主要位于产物的C-2位，这表明在参与反应的两个底物分子中，最初与酶结合的5-氨基酮戊酸分子提供了胆色素原的丙酸“侧链”。产生区域特异性胆色素原的相同酶-[¹⁴C]底物物种可通过与NaBH₄反应而被捕获，这表明最初与酶结合的底物分子是以席夫碱的形式结合的。用5-氨基[5-¹⁴C]酮戊酸进行的常规孵育产生的胆色素原，其标记在C-2和C-11之间均匀分布。因此，人红细胞5-氨基酮戊酸脱水酶的反应机制与我们实验室通过单周转技术研究的其他脱水酶遵循相同的过程。
