Leucine synthesis in chloroplasts: Leucine/isoleucine aminotransferase and valine aminotransferase are different enzymes in spinach chloroplasts

Abstract

Summary This work demonstrates that the complete synthesis of leucine is localized in chloroplasts. Isolated, purified spinach chloroplasts, supplied with 2-oxoisovalerate (3 mmol L −1 ), incorporated [2 −14 C] acetate (2 mmol L −1 ) into leucine as the only amino acid labelled. A leucine/isoleucine aminotransferase and a valine aminotransferase as terminal enzymes of the branched-chain amino acid synthesis (both enzymes belonging to the EC 2.6.1 class) were isolated from chloroplast stroma and purified about 110-fold. The enzymes differed strongly in their substrate specificities. The apparent K m -values for 2-oxoisocaproate and 2-oxo-3-methylvalerate of the leucine/isoleucine aminotransferase and for 2-oxoisovalerate of the valine aminotransferase were 0.12; 0.089 and 1.13 mmol L −1 , respectively. The optimum pH of the aminotransferase reaction was 8. 9. Both enzymes were competitively inhibited by higher concentrations of leucine, isoleucine and valine. However, the efficient regulation of leucine synthesis takes place by a sensitive feedback control of 2-isopropylmalate synthase by leucine at a micromolar level (Hagelstein and Schultz, 1993). Concerning the integration of leucine synthesis into chloroplast metabolism, the problem of pathways competing for the same introductory substrates, acetyl-CoA and 2-oxoisovalerate, is discussed.