Presenting Author Hobart and William Smith Colleges
All living cells are packed with high concentrations of sugars, proteins, and other metabolites. These macromolecules are more likely to interact with enzymes in a crowded cellular environment, thereby influencing the kinetics and thermodynamics of catalysis. In the past, enzyme studies have been conducted under dilute conditions, which is not representative of the environment in living organisms and therefore does not account for the influence of macromolecular crowding. More recently, dextran polymers, which are as large as macromolecules have been used to mimic the crowded cellular environment. These large molecules exclude volume from other molecules thereby influencing the reaction rate in an unpredictable manner. It is believed that that the effects of crowding depend on the rate limiting step (RLS) of enzyme catalysis, since crowding slows diffusion and impedes conformation changes, but also enhances enzyme-substrate binding and increases effective substrate concentration. To test this hypothesis, the effects of crowding on the kinetics of the enzyme glutamate dehydrogenase GDH kinetics were compared for natural substrate glutamate and an alternative substrate norvaline. The RLS of this enzyme-catalyzed reaction with glutamate is the release of the cofactor, NADH, while the slow step with norvaline is the chemistry of catalysis. To more realistically model intracellular conditions, this GDH assay was exposed to bovine serum albumen and partially purified cell lysate, in addition to using the traditional crowding agent, dextran. The rate of the reaction was monitored by absorbance using a spectrophotometer. Repeating the assay at varying temperatures yielded an Eyring plot with thermodynamic data that allowed for the separation of the enthalpy and entropic effects of crowding. Results suggest that excluded volume has a more significant effect than chemical interaction on enzyme kinetics.
