Measuring and comparing activity of enzymes Flashcards
What is a progress curve?
Measures appearance of product (or disappearance of substrate) with time
As more substrate turns into product the amount/concentration of product being made in a certain amount of time will decrease
Enzyme concentration and reaction rate
At different enzyme concentrations the reaction is either faster or slower, if there is a higher concentration of enzymes the rate of reaction increases
Initial velocity is proportional to enzyme concentration when substrate is in excess
Fixed amount of enzyme reaction rate and [substrate]
In a fixed amount of enzyme, when substrate is increased the rate of reaction will increase in a linear way at first but as all active sites become occupied the rate of reaction stops increasing
What is Vmax?
Maximum possible velocity that you can reach with a fixed amount of enzyme, tells us how fast the enzyme can be (max velocity)
What is Km?
substrate concentration at which Vmax is halved, V= Vmax/2 (Michaelis constant), enzyme with a low Km can easily achieve half of its max velocity as only needs very little [substrate]
What is a Lineweaver-Burk plot?
If we plot 1/V vs 1/[S] then we can generate a Lineweaver-Burk plot
This is an inverse plot which has intercepts that tell us what some of these values are hence can calculate the Km and Vmax. The slope (rise/run) of this curve is equal to Km/Vmax
Why is Km important?
Km is important as it characterises one enzyme substrate pair, an enzyme can act on different substrates. It is the substrate concentration needed to reach half Vmax
Low Km= high affinity between enzyme and substrate
High Km= low affinity between enzyme and substrate
Physiological significance of Km
In the cell [S] is often below Km meaning rate control is effective
Physiological substrate concentrations are likely to be found in regions where there are enough active sites available so that substrate molecules are not queuing
What is kcat?
kcat is the number of substrate molecules converted to product per enzyme, per unit time when enzyme is saturated with substrate - helps to define the activity of one enzyme molecule (measure of catalytic activity)
The rate limiting step is k2 - k2 is the turn over rate (kcat) which is defined by the Vmax/[enzyme]
Vmax = kcat * [E]
kcat unit is s-1 (gives the number of times we can turn over a reaction per second)
kcat, Km and catalytic efficiency
A high kcat has the ability to turnover a lot of substrate into product per second
A low Km (low substrate concentration) is required to ‘get up to speed’; high affinity
So kcat/Km gives an overall measure of enzyme efficiency, bigger kcat and smaller Km gives us a higher efficiency for our enzyme
