Measuring and Comparing Activities of Enzymes Flashcards
What do Vo, Vi and Vine signify?
The initial reaction velocity
Describe the reaction profile when the substrate is in excess and the amount of enzyme is increased
Vo is proportional to enzyme concentration, so it is a straight line. As the amount of enzyme increases, the rate of reaction increases.
Describe the reaction profile when there is a fixed amount of enzyme and the amount of substrate increases
Increase in a linear way initially, and then as all the active sites become occupied, it becomes a curve as the rate of reaction stops increasing.
- Vmax is when no more active sites are available
What does Vmax represent?
maximum velocity possible when substrate concentration is infinity. It is specific the the amount of enzyme.
What does Km represent?
The substrate concentration at which V = Vmax/2. Km is also called Michaelis constant
What is the Michaelis-Menten equation and what does it describe?
V = (Vmax[S])/(Km + [S])
describes the V vs. [S] curve
What assumption sis the Michaelis-Menten model based off?
- Product is not converted back to substrate
- Haldanes steady state assumption (that the rate of ES complex formation is equal to the rate of its breakdown)
- Measuring initial rates means that substrate concentration does not change significantly
How do you determine Km, Vmax and Kcat
Plot a graph of 1/V by 1/[S] (it should be linear)
Km: the x intercept = -1/Km
Vmax: the y intercept = 1/Vmax
Kcat: Kcat = Vmax/[E] = K2
What is the importance of Km?
- Characterises one enzyme-substrate pair (if an enzyme can act on different substrates, it will have different Km values for each).
- It is the substrate concentration needed to reach half Vmax.
- If [S] is lower than Km, it means that rate control is effective because substrate is not queuing for the active site. (happens in a cell, shown by linear part of graph)
- can be an indicator for substrate preference
Low Km = high affinity for E+S
High Km = low affinity for that substrate
What is Kcat and how do you measure it?
It is the number of substrate molecules converted to product, per enzyme, per unit of time, wen E is saturated with substrate.
- It is the activity of one enzyme molecule
Kcat = Vmax/[E]
Vmax = Kcat x [E]
How do you determine the overall efficiency of an enzyme?
Kcat/Km
What does a progress curve measure?
It measures the appearance of a produce (or disappearance of substrate) with time
- important to measure the initial reaction velocity for an accurate measurement
what should a peak enzyme have?
high Kcat and low Km
