Abstract C154: Kinetic analysis of the glutaminase activity of L-asparaginases derived from Erwinia chrysanthemi (Erwinase®) and Eshericia coli (Kidrolase).

Abstract

Abstract Glutamine is an essential nutrient for cancer cells and increased glutaminolysis is widely recognised as a key feature of the metabolic reprogramming that occurs during malignant transformation. As a consequence of the role glutamine plays in cancer biology, targeting glutamine metabolism is of considerable interest with inhibition of the mitochondrial enzyme glutaminase showing promise in experimental models. An alternative or adjunct to the strategy of inhibiting glutamine metabolism intracellularly is to deplete extracellular pools of glutamine. L-asparaginases are known to possess glutaminase activity and the purpose of this study is to characterise the kinetics of the glutaminase activity of two clinically approved L-asparaginases, Kidrolase (derived from E. coli) and Erwinase (derived from Erwinia chrysanthemi). Glutaminase activity was measured using a two step reaction: initial reduction of glutamine to glutamate by Kidrolase/Erwinase followed by the NAD dependent conversion of glutamate to ammonia by L-glutamate dehydrogenase (GDH). Each reaction consisted of NAD (2mM), K2PO4 (50mM), GDH (3U), Erwinase or Kidrolase (10U), L-glutamine (concentrations ranged from 5mM to 1.25 μM) in a final volume of 1 ml Tris Acetate buffer (50mM, pH8.6). The reaction was started by the addition of Erwinase/Kidrolase and the formation of NADH monitored for 30 seconds at 340nm using a Cary UV/Vis spectrophotometer and a NADH molar extinction coefficient of 6,220 M-1cm-1. The reaction was linear between glutamine concentrations 100 and 1.25 μM. Using 10U in each reaction, Hanes Woolf kinetic plots generated Vmax parameters of 46.29 and 7.57 μM/min for Erwinase and Kidrolase respectively. Km values were 96.05 and 33.06 μM for Erwinase and Kidrolase respectively. Preliminary studies demonstrate that glutamine depletion induced a dose dependent reduction in cell proliferation in HCT116 p53+/+, HCT116 p53-/-, MDA-MD-231, MDA-MB-453, MCF7, TK10, A549 and T47D cell lines in vitro. In conclusion, these studies demonstrate that Erwinase has significant glutaminase activity and is superior to Kidrolase in terms of glutaminase activity. Erwinase is a good candidate for exploring the efficacy of glutamine depletion strategies and further studies are required to determine whether Erwinase can potentiate the activity of other approaches aimed at restricting the glutamine dependence of tumors. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C154. Citation Format: Roger M. Phillips, Mohammed U. Saleem, Adrian Williams, Jon Morgan. Kinetic analysis of the glutaminase activity of L-asparaginases derived from Erwinia chrysanthemi (Erwinase®) and Eshericia coli (Kidrolase). [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C154.