C-5 Transamination Pathway によるポルフィリン前駆体の生合成に関する研究

Abstract

The formation of δ-aminolevulinate (ALA) is the first step of the biosynthetic pathway of tetrapyrroles, leading to heme, chlorophyll, vitamin B_<12> and other tetrapyrroles. It is generally accepted that ALA is formed by ALA synthase (EC 2.3.1.37) which catalyses the condensation of glycine and succinyl CoA. The enzyme has not been demonstrated in plants. It is thought that the C-5 pathway is the major, possibly the sole, route for the formation of ALA ; a route (C-5 transamination pathway) involving γ, δ-dioxovalerate as an intermediate followed by transamination of ALA has been supported as one of the C-5 pathways. Recently, Varticovski et al. purified and partially characterized alanine : γ, δ-dioxovalerate aminotransferase (AVT) involved in the C-5 transamination pathway, suggesting the possibility that this pathway plays a role in the biosynthesis of ALA in vivo. In the present study, detailed properties of AVT in animals and plants were examined to demonstrate a role of the C-5 transamination pathway in the biosynthesis of ALA. AVT and alanine : glyoxylate aminotransferase (EC 2.6.1.44) were co-purified to homogeneity from bovine liver mitochondria. The ratio of the two activities remained constant during purification and was unchanged by a variety of treatments of the purified enzyme. AVT activity was competitively inhibited by glyoxylate. Some kinetic data were presented. These results show that the two activities are associated with the same protein. The enzyme was much higher in the glyoxylate aminotransferase activity than in the dioxovalerate aminotransferase activity. The purified enzyme had a molecular weight of approximately 200, 000 with four identical subunits and an isoelectric point of 5.4. The activity ratio of AVT to the glyoxylate aminotransferase was determined with alanine : glyoxylate aminotransferase preparations from various mammalian liver and kidney. A specific aminotransferase for L-alanine and γ, δ-dioxovalerate was found in the cucumber seeds. In dark-grown cucumber seedlings, AVT activity in the transitional region between shoot and root was remarkably high compared with that in the cotyledons. The exposure of the dark-grown seedlings to illumination resulted in a rapid and dramatic increase in the activity only in this transitional region. In contrast, the enzyme in the cotyledons, stem, and roots did not respond to illumination. After a 27-h illumination, the enzyme activity in the transitional region was 100-fold higher than that in the cotyledons. Other aminotransferases assayed in the transitional region did not respond to illumination. AVT in the transitional region was also specific for L-alanine and γ, δ-dioxovalerate.