lecture 11 - measuring enzyme activity Flashcards
What do enzyme progress curves measure?
The appearance of product/disappearance of substrate with time
What is the symbol for initial reaction velocity in enzyme catalysis?
Vo
What is the relationship between initial rate of reaction and enzyme concentration when there is excess substrate?
Vo and [E] are directly proportional
What is required for direct proportionality between Vo and [E]?
Excess substrate
What is the relationship between initial rate of reaction and substrate concentration when there is a fixed amount of enzyme?
Initially, there is a linear relationship (first order kinetics) as increased substrate increases the rate. However, eventually all of the enzyme active sites become occupied and the rate stops increasing (zero order kinetics), curve flattens towards asymptote
What are the 2 parts of a Vo vs substrate conc. curve?
First order kinetics (steep linear increase) and then zero order kinetics (curve flattens)
What is Vmax in terms of enzyme catalysis?
The maximum velocity of reaction possible, when substrate concentration is infinite
What is the relative substrate concentration at Vmax?
It’s infinite
What is the symbol for the Michaelis constant?
Km
What is the value of Km?
The substrate concentration at which V=Vmax/2
What is the shape of a Vo vs [S] curve?
Rectangular hyperbola
Wha equation describes the V vs [S] curve?
The Micahelis-Menton equation
What is the Michaelis-Menton equation?
V=(Vmax[S])/(Km+[S])
What are the 3 assumptions made in the Michaelis-Menton equation?
1.) Product is not converted back into substrate, 2.) Steady state: The rate of ES complex formation=the rate of its breakdown into E and P, 3.) As long as there’s excess substrate, [S] does not change significantly at the initial rate
What relationship does a Lineweaver-Burke Plot show?
1/V vs 1/[S]
What is the shape of the curve of a Lineweaver-Burke Plot?
Linear
What is the y-intercept of a Lineweaver-Burke plot?
1/Vmax
What is the x-intercept of a Lineweaver-Burke plot?
-1/Km
Why do enzymes have a different Km value for each substrate they can interact with?
Enzymes will catalyse different reactions at different rates
What is the unit for the Michaelis constant, Km?
concentration unit, e.g. mmol/L, because it is a measure of [S]
What is the equation for Km, in terms of rates of dissociation?
Km ≈ (k-1)/(k1), where k-1=rate of dissociation of S from E and k1= rate of association between E and S (the reverse reaction)
What is the significance of a low Km?
High affinity of E and S
What is the significance of a high Km?
Low affinity of E and S
In a cell, what is the typical range of [S]?
Often below or around the Km value, meaning that rate control is effective and substrates aren’t queuing for active sites
What is the symbol for the turnover number?
Kcat
What does Kcat represent?
The turnover number - the number of substrate molecules converted to product, per enzyme, per unit of time.
What conditions is Kcat measured under?
Enzyme is saturated with substrate
What does the Kcat define?
The catalytic activity of an individual enzyme molecule
What does k2 represent in terms of enzymes?
The rate of production of enzyme and product from ES (enzyme-substrate complex)
What value is Kcat equivalent to?
k2
What is the relationship between Kcat, Vmax and [E]?
Kcat = Vmax/[E]
What are the relative values of Kcat and Km that an enzyme must have to be highly catalytically efficient?
High Kcat, low Km
What ratio shows the efficiency of an enzyme?
Kcat/Km
If Kcat/Km is high, what is the efficiency of an enzyme?
High efficiency
If Kcat/Km is low, what is the efficiency of an enzyme?
Low efficiency
