Examining the effects of crowding on the pre-steady state and the steady state of α-chymotrypsin
Enzymes catalyze many important chemical reactions in biological systems by lowering their activation energy. Studying how enzymes kinetics are affected by concentrated solute molecules and other environmental conditions is crucially important for furthering our understanding of how these bimolecular function in vivo. The cell is a crowded place where the concentration of protein and nucleic acid content is approximately 200-400 g/L (Ellis, 2001). In this project, we sought to study the changes in both pre-steady state and steady state kinetics of α-chymotrypsin as a function of crowding using solutes such as sucrose, betaine and polyethylene glycol (PEG). Examining the pre-steady state kinetics using stopped-flow methods enables us to observe of the effects of crowding on chymotrypsin’s acylation and deacylation steps. The pre-steady state is characterized by a “Burst” that has a different shape and rate than the steady state. We hypothesize that using sucrose or PEG will exhibit uncompetitive inhibition on Chymotrypsin kinetics. We also hypothesize that the Burst phase will have a different rate under crowding conditions. References Ellis, R. J. (2001). Macromolecular crowding: obvious but underappreciated. Trends Biochem. Sci. 26, 597–604.
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