Lab 6: Enzyme Catalysis

Purpose of the lab exercises: Review the function and characteristics of enzymes Become familiar with the spectrophotometer Determine the optimal reaction conditions for the enzyme peroxidase

Chemical Reactions Involves breaking and making bonds Requires an exchange of energy between the molecules and the environment The energy required to start a reaction: ACTIVATION ENERGY (EA) Energy required to break the chemical bonds in the reactants

Enzymes: Activation Energy Decrease the activation energy of a reaction Activation Energy: Energy required to break bonds in reactants Lowering activation energy speeds up reaction

Enzymes: Optimal Conditions Each enzyme has an optimal set of conditions at which the maximum reaction rate occurs Enzymatic reactions are affected by: e.g.: Heat-tolerant bacteria enzymes optimally function at higher temperatures than most enzymes in humans Temperature pH Enzyme conc. Substrate conc.

Chemical Reactions & Enzymes BUT, these things cannot be done in a cell The, internal and external environment of a cell is relatively constant So, how does a cell proceed with reactions? Enzymes!! Catalytic proteins Change the rate of a chemical reaction Not consumed by the reaction

How Enzymes Works The substrate (reactant) moves toward the enzyme’s active site The chemical reaction is triggered by the enzyme The enzyme releases the product

Enzymes Are very specific for their substrate

Enzymes: Catalytic Cycle

Carrying out 4 separate experiments varying: Enzymes: Peroxidase Today’s lab: Determine optimal conditions for the enzyme peroxidase HOW? Carrying out 4 separate experiments varying: TEMPERATURE pH Substrate used in experiments: Mixture of guaiacol (resin from a small tree) + hydrogen peroxide

2,2’ dihydroxy-3,3’ dimethoxybiphenyl (colorless) Peroxidase H3CO OH OCH3 Guaiacol (Yellow) + H2O2 Radical (unstable) 2 H2O 2,2’ dihydroxy-3,3’ dimethoxybiphenyl (colorless) O 3,3’-dimethyoxybipheno-4,4’ quinone (Red) Peroxidase Catalyzes oxidation of guaiacol Transfers hydrogen and electrons from guaiacol to hydrogen peroxide Color change (substrate is yellow and product is red) Monitor progress using a spectrophotometer

Spectrophotometer Measures the amount of light passing through a solution at a specific wavelength Determines how much light was absorbed by a solution

Spectrophotometer In today’s experiments, as the reaction progresses: More and more reddish product forms Absorbance readings will increase

Experiment 1 (Temperature) Enzyme Experiments Four students (two pairs) in each row should work together One pair does Experiments 1 (temperature) The other pair does Experiments 2 (pH) Experiment 1 (Temperature) Experiment 2 (pH)

Lab Summary Need a graph of A500 as a function of time for the temperature experiment Steps: Mark the temperature for each of the four curves in a legend to the graph Calculate from the initial region (straight line portion) of each curve the rate of the reaction at each temperature (A500 per second) and enter these values next to each curve

Lab Summary “Graphing” Mark the temperature clearly for each of the four curves or in a legend to the graph

Lab Summary “Graphing” Calculate rate from the initial region (straight line portion) of each curve at each temperature (A500 per second) and enter these values next to each curve 0.002 A500/sec

Lab Summary “Graphing” The rate of the reaction is linear for about 20 seconds, (straight-line portion” of the reaction) After ~ 20 seconds: There are not enough substrate molecules So the rate slows down Calculate the rate of reaction using only the straight-line portion of the reaction data

Lab Summary “Calculating Rate” Rise Run Rise X2 - X1 Y2 - Y1 = 0.035 A500 Rate = 0.035 A500 15 sec Run 15 sec =0.002 A500/sec

Lab Summary In a similar way, the rate of reaction needs to be calculated for 25oC, 35oC, 45oC and 55oC Temp oC Rate, A500/sec 25 0.0002 35 0.0020 45 0.0015 55 0.0010 On a separate set of coordinate axes, make a second graph relating reaction rate to temperature Label the optimal temperature (the temperature at which the enzyme exhibits its highest rate of activity)

Lab Summary “Graphing” The optimal temperature is 35OC Temp oC Rate, A500/sec 25 0.0002 35 0.0020 45 0.0015 55 0.0010

Lab Summary “Conclusion” At 25oC Molecules are moving more slowly Substrate collides less frequently with enzyme Slower rate of reaction At 45 & 550C The enzyme becomes “denatured” The enzyme no longer functions properly

500 60 sec

A500/sec

Lab Summary Similar analysis of data for the pH experiments should be done

Start with 0 transmittance filter Make sure level set to 340-599nm Start with 0 transmittance Change from transmittance (0) to absorbance on mode (19999) Set wavelength to 500 nm For blank set absorbance to 0

So, let’s get to it!