2. stereocenter

3. Enzyme kinetic assay How fast does the reaction occur?
How good of a catalyst is fumarase?
Rate enhancement?
What factors influence the enzyme activity?
Amount of protein, pH, temperature, concentration of substrate/product, etc.
Need an assay appropriate to the reaction being considered.

4. Bradford assay
Spectral properties of the dye change when bound to protein
More protein: more dye molecules in ‘bound’ form

5. Fumarase assay
Spectral properties of the chemical change reactant (fumarase) vs. product (malate)
Fumarase absorbs ultraviolet light
Carbon-carbon double bond
Malate “doesn’t”
“Follow” the reaction according to the UV absorbance of the reaction solution

6. Follow the reaction At the start: At the end:
100% fumarate
High absorbance
At the end:
Mostly malate
Low absorbance
DAbs/time directly proportional to D[fumarase]/time
Absorbance (l=260nm)
Time (seconds)

7. Rate of reaction How fast does substrate disappear/product appear?
Units: “#molecules/time”
Absorbance depends on concentration:
Determine mM/min
Concentration depends on volume
Calculate mmol/min
More enzyme = faster reaction: normalize to amount of enzyme
Calculate mmol/min/mg

8. Fumarase is a catalyst
How does its catalysis depend on pH (quantitatively)
Learn about the mechanism of catalysis
Identify optimal conditions to measure other variables
ie. optimize pH while you measure effects of temperature, [substrate], etc.
Learn about biology

9. The actual experiment
Mix fumarase (enzyme) with fumarate (sugar/substrate) in a quartz cuvette
Start the reaction!
*Quickly* put cuvette in spec
Spec’s ‘kinetic mode’ measures Dabs/time
Determine initial rate

10. Absorbance
Time

11. A base in the active site is required to ‘activate’ water
A base in the active site is required to ‘activate’ water *improve its nucleophile character*

12. Optimum pH Determine rate (mmol/min/mg) at 4,5,6,7,8,9,10,11
Buffers provided
‘rough’ pH optimum
Make 3 or 4 more buffers to refine the pH optimum

13. Things to think about
How will you make 18 mM sodium phosphate at pH x?
How do you convert change in absorbance over time to change in fumarate (in mmol) over time?