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stereocenter

stereocenter. 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.

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stereocenter

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  1. stereocenter

  2. 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.

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

  4. 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

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

  6. 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

  7. 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

  8. 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

  9. Absorbance Time

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

  11. 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

  12. 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?

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