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Catalytic Characterization of Human Malate Dehydrogenase 1 (MDH1)
Author(s) -
Huynh Ngoc H.,
Hoang An,
Sohl Christal D.
Publication year - 2020
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.2020.34.s1.04837
Subject(s) - nad+ kinase , chemistry , glutathione , biochemistry , enzyme , dehydrogenase , nicotinamide adenine dinucleotide , malate dehydrogenase , citrate synthase , reactive oxygen species , cytosol , enzyme assay , microbiology and biotechnology , biology
Lung cancer is the leading cause of cancer death. Non‐small cell lung cancer (NSCLC) makes up 85% of all types of lung cancer, and includes squamous cancer, adenocarcinomas, and large cell cancer. Malate dehydrogenase (MDH) exists as two forms, MDH1 (cytosolic) and MDH2 (mitochondrial). Both forms are elevated in tumor cells, but only high expression of MDH1 is associated with poor prognosis. Patients with amplified MDH1 have a 50% reduction rate of survival. MDH1 appears to support proliferation of tumor cells by fueling glycolysis with the regeneration of nicotinamide adenine dinucleotide (NAD + ). However, studies of the kinetics of human MDH1 is limited. Our goal is to characterize the catalytic features and regulation of MDH1. We hypothesized that MDH1 activity is inhibited in the presence of reactive oxygen species (ROS). Human MDH1 was expressed heterogenously from Escherichia coli and purified using immobilized cobalt affinity chromatography. The enzyme catalytic activity was determined in the optimal conditions of salt, pH, temperature, and buffer. In the assay, MDH1 activity was calculated from the oxidation rate of NADH to NAD + at 340 nm using a spectrophotometer after incubating MDH1 with oxaloacetate and NADH under steady‐state conditions. To test our hypothesis under oxidative environment, we repeated this assay using oxidized glutathione. The enzyme activity was 6‐fold lower than that measured in reducing conditions. In the absence of the enzyme, oxidized glutathione does not convert NADH to NAD + , which implied the enzyme’s conformation was affected and hence the observed loss in enzyme activity. In the future, we will employ site‐directed mutagenesis to gain an insight whether our observed inhibition was due to the sensitivity of cysteine residues to oxidation. Support or Funding Information This work was funded by a Research Scholar Grant, RSG‐19‐075‐01‐TBE, from the American Cancer Society (C.D.S.), National Institutes of Health R00 CA187594 (C.D.S.), U54CA132384 (SDSU) & U54CA132379 (UC San Diego), MARC 5T34GM008303 (SDSU), and IMSD 5R25GM058906 (SDSU), as well as the California Metabolic Research Foundation (SDSU).