Bio L13&14 Enzyme Kinetics and Assays

Enzyme Assays and Reaction Rates

• Enzyme assays determine enzyme reaction rates experimentally.
• Reaction rate is determined by measuring the change in substrate or product amount over time.
• Enzyme reactions can be monitored with colored substrates or products.
• Spectrophotometers and lab protocols are used to visualize changes in substrate/product.
• Chromogen concentration is determined by measuring absorbance at λ_{max}.
• λ_{max} is used for high sensitivity and to minimize deviations from Beer’s Law.
• Analyte concentration in a sample is determined by plotting a standard curve with known concentrations.
• Enzyme-catalyzed reaction rate corresponds to the slope of the tangent on the progress curve.

Cellulases and Cellobiase Activity

• Cellulases include endoglucanases, exoglucanases, and cellobiases.
• Endoglucanases cut internal bonds in cellulose.
• Exoglucanases remove cellobiose units from cellulose ends.
• Cellobiases hydrolyze cellobiose into two glucose molecules.
• All work together to degrade cellulose into glucose.

Experiment Design for Cellobiase Activity

• Design an experiment to test cellobiase activity in a new fungus species.

Enzyme Extraction

• Reagents: Mushroom, Extraction Buffer [50 mM MOPS Buffer, pH 6].
• Weigh 2g of mushroom and grind with 8ml of Extraction Buffer until a slurry forms.
• Filter the slurry through a filter into a 15 ml falcon tube.
• Transfer 1.5 ml of filtrate into two 2 ml micro-centrifuge tubes and spin to pellet debris.
• Pool the supernatant in a fresh 15 ml falcon tube, label it as cellobiase enzyme extract, and keep it on ice.
• Use 1 ml of enzyme extract for the next part of the experiment.

Enzyme Assay

• Cellobiase breaks down pNPG into glucose and p-nitrophenol.
• P-nitrophenol turns yellow under basic conditions which can be quantified to determine the amount of product.
• A strong base denatures the enzyme and stops the reaction.
• More yellow color indicates more p-nitrophenol, and therefore more substrate breakdown.

Determining Cellobiase Enzymatic Activity

1. Label 8 cuvettes as 1, 3, 4, 5, 6, 9, 12, and 15 min respectively.
2. Pipet 0.5 ml of Stop Solution (200 mM Na2CO3.H_2O, pH 11) into each cuvette.
3. Label one 15 ml centrifuge tube as “Enzyme Assay”. Add 4 ml of Enzyme Substrate solution [0.75 mM p-nitrophenyl glucopyranoside (pNPG)] to this tube.
4. Add 1 ml of the cellobiase enzyme extract to the “Enzyme Assay” tube and start a timer.
5. After 1 min, remove 0.5 ml of the enzyme reaction mix and add it to the cuvette labeled 1 min and mix.
6. Repeat step 5 for all remaining time points.
7. Label one 15 ml centrifuge tube as “Control”. Add 4 ml of Enzyme Substrate solution (0.75 mM p-nitrophenyl glucopyranoside (pNPG)) to this tube.
8. Add 1 ml of Extraction Buffer (MOPS buffer) to the “Control” tube.
9. Add 0.5 ml Stop Solution to a cuvette labeled 0. Remove 0.5 ml from the “Control” tube and add it to the cuvette labeled 0 to be used as the blank.
10. Read the absorbance of all cuvettes using a spectrophotometer at a wavelength of 410 nm with the cuvette labeled 0 min as a blank.
11. Calculate the amount of p-nitrophenol using a standard curve.

Spectrophotometry and Beer's Law

• Spectrophotometer measures absorbance by passing light through a sample and detecting remaining light.
• Beer’s Law: Absorbance is proportional to cell path length and concentration of the analyte.
• Absorbance is a logarithmic measure of light absorbed at a particular wavelength.

Wavelength Selection for Spectrophotometry

• Substances absorb different amounts of light at different wavelengths.
• P-nitrophenol absorbs the most light at 410 nm.
• Using 410 nm provides sensitive measurements and minimizes errors from other compounds.

Standard Curve

• Used to determine unknown amounts of analyte in a sample.
• Uses standards with known analyte concentrations.
• Plot absorbance vs. amount or concentration.
• Use the curve to determine the amount of analyte in an unknown sample.

Measurement of Enzyme Activity

• Use a standard curve to determine the amount of product formed.

Progress Curve and Reaction Rate

• Reaction progress is monitored by measuring product formed over time.
• Initial velocity (Vo) is calculated from the steep part of the curve.

Michaelis-Menten Curve

• Progress curves are determined at different substrate concentrations.
• Initial velocities are plotted against substrate concentrations.

Km and Vmax

• V_{max}.: Maximum reaction velocity at infinite [S].
• Km: Substrate concentration at ½ V{max}, reflects enzyme-substrate affinity.
• High Km means low affinity, low Km means high affinity.

Key Concepts

• Enzyme kinetics are determined empirically using assays.
• Reaction rate is determined by measuring changes in substrate or product amount over time.
• Absorbance is proportional to the path length and chromogen concentration (Beer’s Law).
• The initial rate indicates enzyme activity when the reaction is in a steady state.