Abstract 1832 Dynamics of Malate Dehydrogenase Mutation and Enzyme Activity

Malate dehydrogenase (MDH) is an enzyme that plays a critical role in cellular metabolism. It is found in nearly all living organisms, from bacteria to humans, and is involved in the conversion of malate to oxaloacetate during the citric acid cycle, a key process in energy production (1). It is found in the cytoplasm of prokaryotic cells and in the cytoplasm and mitochondria of eukaryotic cells. In animals, MDH is present in the liver, heart, and skeletal muscles. Additionally, MDH serves as an ideal model system for studying protein folding and dynamics, and it is an important biomarker for various diseases (2). MDH catalyzes the interconversion of malate to oxaloacetate (OAA) while using NAD+/NADH as a cofactor. For this catalytic reaction, a stretch of amino acids defined as the "flexible loop" is important. This "flexible loop" spans amino acids 119-137 in the watermelon glyoxysomal (wgMDH). Two of the active site arginine residues, R124 and R130, fall within the flexible loop region and play important roles in substrate specificity, catalysis, and binding (3). However, the importance of the other loop region residues is unknown. In this study, our main focus was to understand the importance of several other loop residues to get a better insight into the flexible loop region of wgMDH. Site-directed mutagenesis was performed to generate P119W and K135Q wgMDH mutant constructs. Other mutant constructs (M128A/Q, K125Q, R124A, R125Q, D131L, D132N) were obtained from our collaborator. The expression of the wild type (WT) and the mutant wgMDH proteins were induced by Isopropyl β- d-1-thiogalactopyranoside (IPTG) and was purified using Nickel affinity chromatography. Isolated proteins were then run on SDS-PAGE gels to determine the purity, and the concentrations were determined using Bradford assays. Specific activities and Michaelis Menten kinetics of the WT and mutant wgMDH were performed using a stop assay. PyMOL structures of the wgMDH mutants were also studied to understand the effect of mutations in the kinetic parameters compared to the WT-MDH. For M128A, the PyMOL structure showed a shift of the adjacent loop region residues due to the smaller alanine. We hypothesized that this change to alanine will cause a decrease in its enzymatic activity; therefore increasing the Km. The kinetic data that was collected thus far consists of the WT and M128A. The WT kinetic data for differing OAA and NADH concentrations obtained from the plate reader revealed a Vmax of 3018 μM/min and 2257 μM/min respectively. Likewise, the Km values were 76.38 μM and 39.32 μM respectively. Furthermore, M128A kinetic data for differing OAA and NADH concentrations revealed a Vmax of 2073 μM/min and 14270 μM/min respectively. The Km values were 261.8 μM and 444.3 μM respectively. Data collection for the remaining mutants is still ongoing. This study is funded by McNair Scholars Program at SUNY Geneseo, NSF, and SUNY Geneseo Research Foundation.