Lecute 11 Flashcards
Enzymes catalyse thermodynamically favourable reactions by…
Lowering activation energy
What does a progress curve measure?
The appearance of product ( or disappearance of substrate) with time
Spectrophotometetry
A way of measuring compounds in solution
Shape of progress curve
- goes from a linear rate creating product from substrate into a curve
- linear as it just depends on the parameters of the enzyme, - where we do pristine experiments because things aren’t limiting
What is the initial linear part of the curve called?
Initial velocity
- only part that can be used
Velocity of an ezyme refers to
Idealised path in a progress curve
- Measured rate of formation of a product
It is important to measure…
The initial reaction velocity (rate) i.e at time zero- symbol is Vo or Vi or Vinit
Enzyme conc and reaction rate - EXCESS SUBSTATE - will never get to curve of progress curve
- as the amount of enzymes increased, the rate of reaction increases
When substate is not limiting Vo is proportional to…
Enzyme concentration (E)
ONLY WHEN SUBSTRATE IS IN EXCESS
When there is a fixed amount of enzymes as the amount of substate is increased, the rate of reaction:
Increases in a linear way at first but as all active sisters become occupied, the rate of reaction stops increasing
First order kinetic rate
- depends on substrate concentration
Second order kinetics
- rate doesn’t depend on substate concentration
V max =
Maxiumum velocity possible, with a fixed amount of enzyme and unlimited substrate
- how fast it will go with everything ideal?
What is Km
The substate concentration at which V = Vmax/2
Michaelis constant
- related to how well your enzyme binds to substrate - how well it deals with low amounts of substrate
- low km = doesn’t need much to get going at half of it maximum velocity - good at achieving half its maximum velocity
What equation describes the V vs [S]
Michaelis-menten equation
Michelins menten equartion
Many ____ obey the _______________ belabour, this is how we determine their _____ parameters
Many enzymes obey Michaelis-menten behaviour, and this is how we determine their kinetic parameters
Simplifying assumptions of MM model
- product is not converted back to substrate
- holdane stready state assumption: rate of ezyme substrate formation = its rate of breakdown
- by measuring initial rate we assume the substrate conc doesn’t change overtime
How to determine Km and Vmax
- V vs [S] curve is hyperbolic thus hard to directly determine Vmax and Vmax values
- plotting 1/V against 1/[S] gives a straight line
LINEWEAVER-BURK PLOT - intercepts tell us the plots
On the lineweaver-burk plot, the Y-int means
1/Vmax
On the line weaver-burn plot the x-int shows
-1/Km
What does the slope of the lime weaver-burk plot represent
Km/Vmax
What does Km characterise
Km characterises one enzyme-substrate pair (if an enzyme can act on different substrates, it will have a different Km value for each)
Km is the substrate concentration needed to….
Reach half Vmax
Units of Km
Concentration - mol/L
Low Km =
High affinity between enzyme and substrate
High Km =
Low affinity for substrate
Km is appox..
Kd (dissociation constant) - how tightly the enzyme substrates interact
km as an indicator of substrate preference
In the cell, for a particular_________ interaction, __ is often below the _____. This means _____ control is effective.
In the cell, for a particular enzyme-substrate interaction, [S] is often below the Km. This means rate control is effective.
Physiological substrate concentration
- there is enough enzyme sites available so that substate molecules are not cueing up
The turnover number:
Kcat
What is the Kcat?
- the number of substrate molecules converted to product per enzyme, per unit of time, when Enzyme is saturated with substrate
- therefore helps to define the activity of one enzyme molecule - a measure of catalytic activity
If the Michael is-menten model fits:
k2 = Kcat = volume/ [E]
Vmax equation shit
Amount of times we can turn over a reaction in one second
The ‘peak’ enzyme should have:
- a high Kcat (ability to turn over a lot of substrate into product per second)
- a low Km (low substrate concentration requires to ‘get up to speed’: high affinity for the substrate under the Michaelis- Menten assumptions
What is the equation that shows the overall measure of enzyme activity?
Kcat/Km
- higher Kcat over Km, the greater effieicnty of enzyme
Can make Kcat really _____ or Km really _—-
High
Low
What is the fundamental way to quantify the activity of an ezyme?
To meausure the initial reaction rate, Vo at a range of different substrate concentrations
What is the result of Vo at a range of different substrate concentrations and what is it described by.
Results in a hyperbola
- describes by the muchaelis-menten equation
