In vivo and in vitro studies on asparagine biosynthesis in soybean seedlings

Abstract

The biosynthesis of asparagine in plants was investigated by feeding radioactive metabolites to soybean cotyledons and by extracting an asparagine synthetase from the same tissue. Soybean cotyledon slices were supplied with radioactive succinate, malate, or aspartate in the presence or absence of various unlabeled metabolites for periods of up to 80 min. Neither aspartate nor malate was rapidly converted to asparagine; labeled aspartate was converted largely to malate. Labeled succinate was rapidly converted to asparagine, and several lines of evidence suggested that fumarate, malate, and aspartate are intermediates. The results suggest that asparagine biosynthesis in plant cells is compartmentalized beginning with succinate. Although results were also consistent with asparagine formation via aspartate, metabolism of a mixture of [ 14C] plus [ 3H]succinate resulted in a lower 14 C 3 H ratio in asparagine than aspartate, suggesting that some asparagine may be formed via another pathway. Demonstration of a glutamine-linked asparagine synthetase in soybean cotyledons supports the idea that asparagine is formed via aspartate. The enzyme requires aspartate ( K m = 2.2 m m), glutamine ( K m = 0.12 m m), ATP ( K m = 0.066 m m), magnesium ion, and sulfhydryl protection. It has a pH optimum of 7.7 and is not located in mitochrondria. A small amount of asparagine was formed when ammonium ion was substituted for glutamine, but the K m of the enzyme for ammonium ion was about 25-fold greater than the K m for glutamine suggesting that glutamine is the physiologically important substrate. Soybean cotyledons actively convert [ 14C]-cyanide to asparagine, apparently via β-cyanoalanine. However, malate was also rapidly labeled from [ 14C]cyanide and this result cannot be explained by known metabolic pathways.