FIII: Enzyme Kinetics (Slides 20 - 67) Flashcards
What do enzymes do?
Speed up the rate of reaction proportionally to the amount of enzyme that is present.
Enzyme Assay
The process of measuring enzyme catalyzed reaction rates
Enzyme Kinetics
The mathematical analysis on how rate varies when you increase substrate concentration, but keep enzyme concentration constant.
What happens when an enzyme acts on a substrate?
The substrate decreases and the product increases.
What would chemists use instead of the real enzyme reaction products?
Direct analysis of enzyme reaction products can be time consuming - a better way is using artificial substrates
What is an artificial substrate?
It is a molecular “look alike” for the real substrate
What does Trypsin hydrolyze?
Trypsin hydrolyzes the C-terminal of Arg and Lys in a peptide
How can you measure the artificial enzyme?
The artificial enzyme will have a colour, and you can directly measure the concentration of any molecule with a colour.
What can some natural substrates show after conversion to product (think NADH and NAD+)
Some substrates can show absorbing changes
How does NADH show an absorbance change?
Before getting oxidized, NADH absorbs light at the 340nM wavelength. Once oxidized to NAD+, it does not absorb light at that wavelength which can be used as a progress tracker and a way of measuring rate of reaction
What happens to absorbance as substrate is converted into product?
Absorbance decreases.
What are chromophores?
Conjugated double bonds
What is a conjugated double bond?
The double bond is on every other bond (think aromatics)
What is the wavelength of light a human can visibly see?
400-700 nM
What wavelength can coloured compounds absorb?
400 - 700nM
What wavelength of light can naturally biochemical chromophores absorb?
Between 200 - 400nM (much shorter)
What type of enzyme is NADH
An oxidoreductase, also a chromophore
What wavelength do larger chromophores absorb at?
Longer wavelength
What machine measures absorbance?
A spectrophotometer (also known as a microplane reader), which will give you absorbance reading.
What units does absorbance have?
Unitless, its a ratio: log(Io/l) therefore the units cancel out
What does the Beer-Lambert Law Suggest?
That absorbance is proportional to sample concentration and sample thickness
What is the Beer-Lamber Law?
A = e x c x l
e = molar extinction coefficent (mol l cm)
c = sample concentration (mol L)
l = sample thickness (cm)
*Absorbance is always unites
What is the Rate of Reaction defined as?
The change in [substrate] or [product] per unit time
- Measured in mol/L/min
What is the formula for Rate of reaction 1.)
1.) [Concentration] of Substrate Used/Time
2.) [Concentration] of Product Formed/Time
