Abstract 2014 A Comprehensive Analysis of Key Metabolic Regulators of Malate Dehydrogenase Influencing Mitochondrial Metabolism
Year: 2024
Type: conference-abstract
Abstract: Expressed in nearly all cell types, malate dehydrogenase (MDH) is a critical component of cellular metabolism. MDH is a driving force behind the conversion of malate to oxaloacetate (OAA), a critical component in the citric acid cycle, in-turn generating ATP for energy. Depending on the organism and cell type studied, there are multiple isoforms and splice variants of MDH. In this study we focus on human mitochondrial MDH (hMDH2). hMDH2 integrates in many cellular homeostasis pathways. hMDH2 is regulated by various metabolites via allosteric regulation, substrate regulation and feedback inhibition. Numerous studies have investigated the inhibition of MDH by various metabolites; however, these investigations have been conducted independently, lacking a standardized method for direct comparison. This study addresses this gap, focusing on hMDH2 activity regulation in the context of the tricarboxylic acid (TCA) metabolism Following an evaluation of known inhibitors listed in the Brenda Enzyme Database, the impact of diverse metabolites on hMDH2 activity was systematically examined. Metabolites such as ATP, ADP, AMP, NAD+, αKG, pyruvate, succinate, aspartate, glutamate, glutamine, fumarate, Acetyl Coenzyme A, citrate, isocitrate, and malate were considered. hMDH2 activity was measured with a real-time method using a 96-well format. Enzyme concentration required to achieve a linear rate was first determined, followed up by determination of hMDH2 activity using OAA to initiate the reaction. The reduction of OAA to Malate was observed with oxidation of NADH to NAD+ at 340nm. A range of inhibitors ranging from 100mM to 0.001mM and no inhibitor was used to determine dynamic range adjustment, avoid saturation, improve reproducibility, and optimize assay conditions. In conclusion, the dataset derived from plate reader assays and the determination of IC50 values allows for a direct comparison of potential hMDH2 inhibitors. Additionally, this study delves into the conformational changes induced by select metabolites on wild-type MDH using Small-Angle X-ray Scattering (SAXS) analysis. By comparing SAXS profiles with and without metabolites, the research aims to deepen our understanding of how regulatory molecules modulate MDH's conformation, oligomeric state, and dynamics. These insights into the nature of metabolite regulation of MDH, combined with structure-activity relationships, are crucial for optimizing lead compounds and advancing targeted drug development. This knowledge holds particular significance in diseases where MDH dysregulation is a contributing factor, offering potential therapeutic interventions and contributing to the advancement of our understanding in this critical area of cellular metabolism. This work was supported in part by the NSF MCB RUI-1932755. This work was also conducted at the Advanced Light Source (ALS), on behalf of the Department of Energy, Office of Basic Energy Sciences, through the Integrated Diffraction Analysis Technologies (IDAT) program.
Source: Journal of Biological Chemistry
Institution: University of San Diego
Language: English
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: gold
Estimated APC paid: $2,800
OpenAlex ID: W4393166545