Stereocenter. stereocenter Enzyme kinetic assay How fast does the reaction occur? How good of a catalyst is fumarase? Rate enhancement? What factors.

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Presentation transcript:

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. Need an assay appropriate to the reaction being considered.

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

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

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)

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

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

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

Absorbance Time

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*

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

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?