Location: Exhibit/Poster Hall A-B - Pennsylvania Convention Center
Poster Board Number: A173
Kristin Slade (Hobart and William Smith Colleges), Andrea Desrochers (Hobart and William Smith Colleges), Emily Rundlett (Hobart and William Smith Colleges), Maddie Schleicher (Hobart and William Smith Colleges)
Presenting Author Hobart and William Smith Colleges
Most of our understanding of enzyme kinetics is based on experiments conducted in dilute solution, which does not accurately represent crowded cellular environments. Cells contain high concentrations (300-400g/L) of various macromolecules such as proteins, carbohydrates and ribosomes. This crowding reduces the volume of solvent available for other molecules in the solution and has previously been shown to impact the behavior of enzymes. To study these potential consequences, the Michaelis-Menten kinetics of glutamate dehydrogenase (GDH) was monitored in the presence of high concentrations of “crowding agents” such as dextran, a glucose polymer. Given this enzyme’s central role at interface of nitrogen and carbohydrate metabolism, GDH is highly regulated. Our results suggest that crowding may influence this regulation. For example, leucine is a less effective activator of GDH in the presence of dextran. Furthermore, crowding enhances substrate inhibition of GDH in a pH-dependent manner. The resulting effects from crowding were substrate dependent such that the presence of dextran decreased the Vmax more with the NADP+ cofactor than with NAD+. Both dextran and BSA decreased the Km of NADP+ but did not affect NAD+. The effects of crowding on the enthalpy (ΔH҂) and entropy (ΔS҂) of activations determined from Eyring plots also were also substrate and pH-dependent. Overall, these results highlight the importance of studying enzymes especially heavily regulated ones like GDH under crowded conditions that more realistically mimic the crowded cellular environment.
Macromolecular crowding alters substrate inhibition of glutamate dehydrogenase (GDH) in a pH-dependent manner. The kinetic assays were run in 0.1 M pyrophosphate buffer with 1 µM EDTA. Final concentrations were 1.6 g/L for NAD+, 0.02 g/L for GDH, and 240 g/L for glucose or dextran 150.
