Final answer:
To determine Km and Vmax, experimentally measure reaction velocities at different substrate levels, plot them to get a Michaelis-Menten plot, or use a Lineweaver-Burk plot to find the intercepts, from which Km and Vmax can be calculated. Enzyme concentration is crucial for these calculations, as well as determining the turnover number and catalytic efficiency.
Step-by-step explanation:
To experimentally determine Km and Vmax of an enzyme, one would conduct an experiment measuring the reaction rate (velocity) at various substrate concentrations. First, using a graphing program, plot the reaction velocity versus substrate concentration ([S]) to create a Michaelis-Menten plot. If the graphing program can fit a hyperbolic equation, it can be used to directly determine Km and Vmax. Next, construct a Lineweaver-Burk plot, which is a double reciprocal graph (1/v versus 1/[S]). Fit the data to a straight line to calculate Km and Vmax. The slope of this line will be Km/Vmax, and the y-intercept will be 1/Vmax. The x-intercept of the plot is -1/Km. These calculations are based on the enzyme concentration in the cuvette, which can be calculated knowing the superstock concentration of the enzyme. Finally, determine the turnover number (kcat) and the measure of catalytic efficiency by calculating the ratio of kcat/Km.