Elucidation of multifunctionality and substrate specificity of human aspartate aminotransferases

Abstract

d-Serine and d-aspartate play crucial physiological roles in mammals. d-Serine is produced by serine racemase, but the biosynthetic pathway of d-aspartate remains unclear. In this study, we investigated the substrate specificity and multifunctionality of human aspartate aminotransferases (hGOT1 and hGOT2) to reveal whether they possess d-Amino acid metabolic activity. Neither enzyme displayed racemase activity toward various amino acids including aspartate, although slight alanine racemase activity was detected. Likewise, neither exhibited lyase, dehydratase, or aspartate decarboxylase activities. Regarding aminotransferase activity, both displayed high activity toward l-aspartate and l-glutamate as amino donors, and they acted on some l-Amino acids, but not d-Amino acids. Intriguingly, we found that aminotransferase activity for oxaloacetate followed sigmoidal kinetics rather than typical Michaelis-Menten kinetics. Thermal shift assay experiments suggested that pyridoxal-5′-phosphate and oxaloacetate are involved in protein stability and the ability to bind oxaloacetate is different from hGOT1 and hGOT2. In summary, hGOTs accept some amino donors and acceptors and unique capacity to bind oxaloacetate.