Phosphomimics of Cytosolic Human Malate Dehydrogenase 1 Alters Enzymatic Activity
Grayzel, Alexander
Grayzel, Alexander
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Abstract
Cytosolic human malate dehydrogenase 1 (hMDH1) plays a central role in cellular metabolism by catalyzing the reversible conversion of malate to oxaloacetate using NAD?/NADH in the malate-aspartate shuttle. A predicted phosphorylation site within the conserved His-Ser-Ser-Thr (HSST) motif (residues 187-190) suggests a potential regulatory mechanism affecting enzymatic activity. This study investigates the functional consequences of phosphorylation at serine residues 188 and 189 using phosphomimetic mutations. Site-directed mutagenesis was employed to generate S188D and S189D variants of hMDH1, mimicking phosphorylation. Recombinant proteins were expressed, purified via Ni-affinity chromatography, and assessed for purity using SDS-PAGE. Enzymatic activity was evaluated using Michaelis-Menten kinetics by measuring NADH production across increasing substrate concentrations. Kinetic analysis demonstrated that the mutations differentially affect enzyme function. The S188D variant showed a lower Km compared to wild-type hMDH1, indicating increased affinity for NADH, but exhibited a reduced Vmax, suggesting decreased catalytic efficiency. In contrast, the S189D variant displayed a higher Km and increased Vmax, indicating reduced substrate affinity but enhanced catalytic turnover. These results indicate that phosphorylation at adjacent residues within the HSST motif can distinctly modulate hMDH1 activity. This study highlights the importance of site-specific post-translational modifications in regulating metabolic enzymes and suggests a mechanism by which hMDH1 activity may be fine-tuned.
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Date
1/1/2026
Student Status
Senior (Graduating in 2026)
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Poster
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Biochemistry
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College of Arts and Sciences
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Life Science