Glyoxylate aminotransferases and ureide catabolism in the developing fruits of legumes

Abstract

The metabolism of glyoxylate released as a result of ureide catabolism in the developing fruits of legumes was examined through an investigation of alanine-, glutamate- and serine-glyoxylate aminotransferase activity in these tissues. A study of the distribution of these activities among the tissues of the developing fruits of two ureide-transporting legumes, French bean (Phaseolus vulgaris) and cowpea (Vigna unguiculata) and an amide-transporter, snow pea (Pisum sativum), was conducted. Serine-glyoxylate aminotransferase activity in each species was very low relative to the alanine- and glutamate-glyoxylate aminotransferase activity. In all three species most of the aminotransferase activity was in the pods. In cowpea, alanine- and glutamate-glyoxylate aminotransferase activity was high in the embryos relative to the seed coats, whereas in French bean and snow pea the seed coats had higher activity than the embryos. Three glyoxylate aminotransferases were isolated and partially purified from extracts of French bean and snow pea developing fruits. These were a serine-glyoxylate aminotransferase (SGAT), which primarily catalyzes the serine glyoxylate aminotransferase reaction; a glutamate-glyoxylate aminotransferase (GGAT), which catalyzes both the glutamate- and alanine-glyoxylate aminotransferase reactions; and an alanine-glyoxylate aminotransferase (AGAT), which catalyzes the alanine-glyoxylate aminotransferase reaction. Sucrose-density gradient ultracentrifugation indicated that the SGAT and GGAT from French bean fruits are located in the peroxisomes, and that the AGAT is located outside the peroxisomes, probably in the mitochondria or chloroplasts. These results confirm the existence of an alanine-glyoxylate aminotransferase (AGAT) as an enzyme distinct from the GGAT. The results of initial velocity kinetic studies of the enzymes isolated from French bean and snow pea, conducted at pH 7.1, were consistent with a Ping Pong reaction mechanism involving pyridoxal phosphate as a co-enzyme. The Km values obtained for the substrates of the enzymes were in all cases higher for the amino acid than for glyoxylate. For the French bean enzymes the Km for the amino acids was about ten-fold higher than that for glyxoylate. The affinity of the snow pea enzymes for glyoxylate was less than that of the French bean enzymes. Alanine--ketoglutarate aminotransferase activity was associated with the GGAT and this activity was reversible, in contrast to the alanine- and glutamate-glyoxylate aminotransferase activity of this enzyme. The reversible aspartate--ketoglutarate aminotransferase activity of the French bean developing fruits was not catalyzed by the glyoxylate aminotransferases. The pH optimum of the GGAT and the SGAT from French bean was approximately 8.3. The molecular weight of the GGAT and AGAT from French bean estimated by gel filtration was 104 kDa. The results are discussed in the context of the metabolic pathway of ureide catabolism, and its location within the fruits of different ureide transporting legumes.