Enzyme Catalysis Lab: • Measuring the RATE of an enzyme catalyzed reaction: 2H2O2 2H2O +O2. • What is an enzyme? Structure, function, inhibition etc. • Catalase role in cell? • H2O2 to H2O + O2 is spontaneous but SLOW! • What is free energy and how relates to reaction being spontaneous or not? • Catalysts allow cells to carry out reactions at LOWER temperatures suitable for a cell ( high temps usually denature proteins). • E+S ES (a complex) E+P • Substrate: H2O2 • Enzyme: Catalase • Stopping reagent: H2SO4 (denatures enzyme) • Aim: To see how much H2O2 LEFT after a set time. • Titrated against KMnO4. • Reaction: H2O2 + KMnO4 +H2SO4 K2SO4 + MnSO4 + H2O + O2 • H2O2 reactant and limited amount. • K2SO4 and MnSO4 colorless and formed until all available H2O2 converted. • When no more H2O2 available the brown/purple color of KMnO4 remains (just before this point is the “endpoint”) • As run enzyme reaction longer, LESS H2O2 remaining, so LESS KMnO4 used before brown/purple color remains. • Therefore amount of KMnO4 used is proportional to amount of H2O2 left after reaction stopped. • Baseline – need to know how much H2O2 INITIALLY in the 1.5% solution of H2O2. • Do all the steps EXCEPT adding catalase. • Titrate against KMnO4 and amount of KMnO4 used = amount of H2O2 in the 1.5% H2O2 solution initially = baseline amount. • Final reading = 15ml, Initial reading = 11ml. Amount of H2O2 originally in solution = 15-11 = 4ml (baseline amount) • To find out amount of H2O2 USED at each time point = baseline amount (starting amount) minus the amount LEFT at each time point. • So, amount H2O2 used at 5min = 4ml – 1ml left = 3ml USED UP after 5min of the reaction started. • Study table in lab. • Independent variable = what is changing (Time/min) • Dependent variable = how measure change over time (amount of H2O2 USED UP/ml) • Rate = deltaY divided by delta X. • Know what the different graphs telling you: Draw the graphs for: If enzyme amount set and substrate amount set then what kind of graph if plot product amount substrate converted vs time? If enzyme amount set and increasing substrate concentration what graph would you get if plot amount of substrate converted vs time If substrate amount set and increasing enzyme concentration what graph would you get if plot amount of substrate converted vs time?