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
What is Enzyme Activity measuring?
The amount of enzyme present
What is the formula for Enzyme Activity?
Moles of Substrate/Time or Moles of Product/Time
*Can also be demonstrated by rate x volume
What are the units of enzyme activity
umol/minute (1 enzyme unit)
What is Specific Activity?
Measuring the purity of the enzyme during purification.
What is the Specific Activity measuring
The number of enzyme per milligram of total protein.
Formula for Specific Activity?
Enzyme Activity (umol/min)/Crude Protein(mg)
What is the formula for Molar Activity?
The Specific Activity x Molar Mass of Enzyme
What is Molar activity equal to?
The “turnover number” - The Number of Catalytic reactions per enzyme per second.
What is a progress curve?
A post of substrate or product concentration over a period of time
Where would the initial velocity be taken from?
The slope of the curve at time 0
When you measure substrate concentration what is the slope?
-slope
When you measure product concentration what is the slope
+slope
What is your initial rate (Vo) equal to?
k2[ES]
What does putting the initial rate of a function of time = 0 demonstrate?
The elimination of the reverse reaction.
What is Vmax?
Vmax is the theoretical maximum velocity for enzyme concentration`
What is Km
The Michaelis constant. Half of the maximum velocity given for substrate concentration
What is Vmax?
The “upper limit”, where substrate concentration can increase but your rate can not increase (a plateau)
What does Vmax indicate?
The catalytic rate: where 100% of the enzyme is occupied by the substrate.
What does a higher Vmax mean
A faster reaction, better catalysis
Why is Vmax considered a “pseudo” constant?
It is only a constant if the amount of enzyme is fixed
- if enzyme concentration increases, the Vmax will increase.
What is K2?
K2 is a true constant, and is the turnover number (molar activity) of the enzyme
