Role of aminotransferases in glutamate metabolism of human

Human erythrocytes require a continual supply of glutamate to support glutathione synthesis, but are unable to transport this amino acid across their cell mem- brane. Consequently, erythrocytes rely on de novo gluta- mate biosynthesis from a-ketoglutarate and glutamine to maintain intracellular levels of glutamate. Erythrocytic glutamate biosynthesis is catalyzed by three enzymes, alanine aminotransferase (ALT), aspartate aminotransfer- ase (AST), and glutamine aminohydrolase (GA). Although the presence of these enzymes in RBCs has been well documented, the relative contributions of each pathway have not been established. Understanding the relative contributions of each biosynthetic pathway is critical for designing effective therapies for sickle cell disease, hemolytic anemia, pulmonary hypertension, and other glutathione-related disorders. In this study, we use multi- dimensional 1 H- 13 C nuclear magnetic resonance (NMR) spectroscopy and multiple reaction mode mass spectrom- etry (MRM-MS) to measure the kinetics of de novo glu- tamate biosynthesis via AST, ALT, and GA in intact cells and RBC lysates. We show that up to 89% of the eryth- rocyte glutamate pool can be derived from ALT and that ALT-derived glutamate is subsequently used for glutathi- one synthesis.