Development of fully automated isocitrate dehydrogenase enzyme activity assay kit and recombinant production of isocitrate dehidrogenase enzyme

Isocitrate dehydrogenase (IDH) is an enzyme that catalyzes the production of α-ketoglutarate (α-KG) and carbon dioxide (CO2) by performing the oxidative decarboxylation of isocitrate. It is known that this enzyme, which is intensely found in many tissue, acts as a source of nicotinamide adenine dinucleotide phosphate (NADPH + H) and plays a crucial role in glucose, fatty acid and glutamine metabolism. The NADPH + H molecule has an important role in both cellular biosynthesis reactions and in defense against oxidants due to its use as a reducing equivalent molecule. Mutations in the IDH enzyme lead to neomorphic activity in the enzyme, causing it to use α-KG, which normally occurs as a product, as a substrate and consume it contrary to the production of NADPH + H. This change causes cancer development by affecting many pathways at the cellular level especially in gliomas; IDH mutation has also been reported in leukemia, lymphoma, cholangiocarcinoma, chondrosarcoma and pancreatic tumors. It is also known that increased NADP + -dependent IDH activity in serum gives more sensitive results than aspartate amino transferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), γ-glutamyl transferase (GGT) enzymes, which are routinely used in the determination of liver tissue damage. There is no simple and inexpensive method that can detect changes in the activity of this clinically important enzyme and its mutant form. The methods used in investigating on mutant enzymes are techniques that require intervention and work with tissue samples taken by biopsy. With this study, it is planned to develop a kit that can be used in automated chemistry analyzers which are based in all routine biochemistry laboratories. It is intended to measure the NADP+ -dependent normal and mutant IDH enzyme activity in blood samples that had already been taken from patients. It is aimed to have information about enzyme activity using spectrophotometric method by tracking the absorbance change at 340 nm of NADPH+H molecule, which is a molecule used by the enzyme as a cofactor. It is aimed to produce recombinant mutant and normal form of NADP + -dependent IDH enzymes that can be used as calibrator-control material to be used in the kit studies to be developed. During the course of the study, it was determined that the activity of the mutant enzyme was not suitable for measurement as initially planned. With the development of the NADP+ -dependent IDH activity measurement method, a linear measurement kit with a CV % value of less than 2, LoB 0.1744 U / L performance characteristics between 0 - 1200 U / L was produced. Recombinant production of both normal form and R132H mutant form NADP+ -dependent IDH enzymes was performed in Pichia Pastoris yeast cells, an eukaryotic microorganism. Vmax and KM values of recombinant normal NADP+-dependent IDH enzyme for its substrate were found as 4.55 μM / min and 108 μM, respectively. Keywords: Isocitrate dehydrogenase, enzyme activity measurement, recombinant, Pichia Pastori