Glutamate biosynthesis by Clostridium kluyveri: An unusual distribution of radioactivity in glutamic acid

Abstract

1. 1. Cell-free extracts prepared from dried cells of Clostridium kluyveri were found to contain citrate synthase (EC 4.1.3.7), aconitase (EC 4.2.1.3), isocitrate dehydrogenase (EC 1.1.1.42) and glutamate dehydrogenase (EC 1.4.1.3). Biosynthesis of glutamic acid is thus possible via the normal citric acid cycle reactions. 2. 2. Maximal activity of citrate synthase and aconitase, but not isocitrate and glutamate dehydrogenases was dependent upon preparation of the cell extracts under reducing conditions. Isocitrate dehydrogenase was dependent on NADP + and Mn 2+ whilst glutamate dehydrogenase was dependent on either NADH or NADPH. 3. 3. Glutamic acid, synthesised by cell extracts from [5- 14C]citric acid, was degraded and found to contain 92% of the radioactivity in the α-carboxyl position. When [1,5- 14C]citric acid was the precursor of glutamic acid, the distribution of radioactivity was 48% in the α-carboxyl group and 46% in the remainder of the molecule. The stereospecificity of aconitase therefore appeared to be normal. 4. 4. Degradation of glutamic acid, synthesised from [4- 14C]aspartic acid, by extracts prepared from cells grown in 1964 yielded the following results: 47% of the label was in the α-carboxyl and 51% in the γ-aminobutyrate moiety of glutamic acid. In the case of cells grown in 1966 the distribution was 51% in the α-carboxyl and 39% in the γ-aminobutyrate. When [1- 14C]acetate was the substrate the following distribution was obtained (1966 cells): 59% in the α-carboxyl and 25% in the γ-carboxyl. These results indicate (a) an unusual stereospecificity in the citrate synthase and/or (b) the presence of unknown reactions which affect the distribution of the radioactivity in the glutamic acid.