Presentation Type
Poster
LARGE-SCALE PRODUCTION, BIOCHEMICAL AND STRUCTURAL CHARACTERIZATION OF LACTATE DEHYDROGENASE
Abstract
Lactate dehydrogenase (LDH) catalyzes the interconversion of pyruvate and lactate in the final step of anaerobic glycolysis. Many invasive tumors depend on an increased rate of glycolysis in order to maximize their growth potential under hypoxic environments, a phenomenon known as the “Warburg Effect.” By finding a way to inhibit LDH, contributions would be made towards the management of cancerous cells, by inhibition of their energy production.
Human LDH isoenzyme A was subcloned into an in-house vector (pET28a-PreScission), and overexpressed in E. Coli Tuner(DE3) cells as a fusion protein with Maltose Binding Protein to increase solubility. The fusion protein was cleaved, purified, and subjected to crystallization trials using vapor diffusion.
Crystals of the binary complex of LDH-A with NADH were obtained and conditions are being optimized to increase crystal size and diffraction quality. Steady-state kinetic characterization revealed Km(Pyruvate) to be 130 ± 19 µM and the specific activity to be 382 ± 16 U/mg. The IC50 value for the inhibitor Oxamate was determined to be 65 ± 2 µM.
Future plans include high-throughput screening (HTS) for novel LDH-A inhibitors, co-crystallization of LDH-A with HTS hit compounds and the structure-based design of potent and specific inhibitors of this enzyme.
Categories
Biomedical Sciences
Research Type
Thesis
Mentor Information
Dr. Ernst Schönbrunn
LARGE-SCALE PRODUCTION, BIOCHEMICAL AND STRUCTURAL CHARACTERIZATION OF LACTATE DEHYDROGENASE
Lactate dehydrogenase (LDH) catalyzes the interconversion of pyruvate and lactate in the final step of anaerobic glycolysis. Many invasive tumors depend on an increased rate of glycolysis in order to maximize their growth potential under hypoxic environments, a phenomenon known as the “Warburg Effect.” By finding a way to inhibit LDH, contributions would be made towards the management of cancerous cells, by inhibition of their energy production.
Human LDH isoenzyme A was subcloned into an in-house vector (pET28a-PreScission), and overexpressed in E. Coli Tuner(DE3) cells as a fusion protein with Maltose Binding Protein to increase solubility. The fusion protein was cleaved, purified, and subjected to crystallization trials using vapor diffusion.
Crystals of the binary complex of LDH-A with NADH were obtained and conditions are being optimized to increase crystal size and diffraction quality. Steady-state kinetic characterization revealed Km(Pyruvate) to be 130 ± 19 µM and the specific activity to be 382 ± 16 U/mg. The IC50 value for the inhibitor Oxamate was determined to be 65 ± 2 µM.
Future plans include high-throughput screening (HTS) for novel LDH-A inhibitors, co-crystallization of LDH-A with HTS hit compounds and the structure-based design of potent and specific inhibitors of this enzyme.
