Here, for the first time, we report an NMR spectroscopy study of L-Glutamine (Gln) conversion by Glutaminase (Glnase), which shows that the reaction involves two distinct steps. In the first step, Glnase rapidly hydrolyzes Gin to Glutamate (Glu) (similar to 16.87 mu mol of Gln/min/mg of Glnase) and in the second step, Glu generated in the first step is decarboxylated into gamma-amino butyric acid (GABA) with a much slower rate (similar to 0.185 mu mol/min/mg). When Glnase was added to the sample containing L-Glu alone, it was also converted to GABA, at a similar rate as in the second step mentioned above. The rate of Glu decarboxylation into GABA by Glnase is about an order of magnitude lower than that by commonly known enzyme, Glutamate decarboxylase. Potential impact of these findings, on the mechanistic aspects of Gln-Glu shuttle in neuroscience and glutaminolysis in tumors, is discussed. (C) 2014 Elsevier Inc. All rights reserved.