L4 - Enzyme Kinetics Flashcards
Define the transition state
The high energy intermediate that lies between the substrate and the product
Define the activation energy
The minimum energy the substrate must have to allow the reaction
What are enzymes?
Biological catalysts that increase the rate of reaction by lowering the activation energy.
They do this by facilitating the formation of the transition state
What are important features of enzymes?
Specific Unchanged after the reaction DO NOT AFFECT THE REACTION EQUILIBRIUM Increase the rate of reaction Protein (with one or two exceptions) May require associated cofactors
Why are we interested in enzymes?
Inheritable genetic disorders
Overactive enzymes can cause disease
Measurement of enzyme activity for diagnosis
Inhibition of enzymes by drugs
Define an active site
A place in the enzyme where substrates bind and where chemical reactions occur
What are the features of an active site?
- A small part of the enzyme - few aa
- Formed from aa from different parts of the sequence
- Are clefts or crevices - usually exludes water
- Complimentary shape to substrate - lock and key hypothesis
What is the lock and key hypothesis?
Active sites have a complimentary shape to the substrate
What is the induced fit hypothesis?
Binding of the substrate can induce changes in conformation. Therefore the active site only forms a complimentary shape after binding of the substrate
How does an enzyme bind its substrate?
Not too tightly with multiple non-covalent bonds (any type)
Explain the process through which enzymes work
- S alone
- E binds the substrate to form ES complex (lower E)
- ES transition state occurs (high energy state)
- P formed, alone
What is Vo?
The initial rate of reaction. This is the only time during the reaction where the concentration of substrate is known
What is the shape of a graph that plots reaction velocity (y axis) against substrate concentration (x axis)?
Rectangular hyperbola - as the enzyme-catalysed reaction reaches a maximum velocity and plateaus
What does the Michaelis-Menten model for enzyme catalysis propose?
That a specific complex between the enzyme and the substrate is a necessary intermediate in catalysis.
It predicts that a plot of Vo versus [S] will be a rectangular hyperbola.
NOT ALL ENZYMES OBEY THIS MODEL e.g. those that follow cooperativity like haemoglobin
What does Km and Vmax mean?
Km = Substrate concentration that gives half maximal velocity Vmax = Maximum rate when all enzymes in active sites are saturated with substrate (this will vary depending on the concentration of the enzyme)
What does a high Km or low Km tell you about the enzyme and its substrate?
High Km - enzyme has a low affinity for its substrate
Low Km - enzyme has a high affinity for its substrate
Hexokinase and glucokinase are enzyme which catalyse the same reaction (glucose -> glucose-6-phosphate). What is the difference between them?
Glucokinase (present in liver) has a higher Km than hexokinase (in all tissues), therefore glucokinase is only active at high concentrations of glucose - switching on glycolysis in the liver after feeding
The Michaelis-Menten equation can be rearranged to give a linear plot. What is its name?
The Lineweaver-Burk plot
In the Lineweaver Burk plot. What is the the y axis and x axis intercepts give you?
Y axis: 1/Vmax
X axis: -1/Km
What are enzyme inhibitors?
Molecules that slow down or prevent an enzyme reaction
What types of enzyme inhibitors are there?
- Irreversible - generally form covalent bonds
- Reversible - non-covalent, can freely dissociate
(i) Competitive
(ii) Non-competitive
What are competitive inhibitors and how do they affect a reaction?
Resemble the substrate and bind to the active site of enzyme, reducing the proportion of enzyme bound to the substrate. Affect Km but not Vmax
What are non-competitive inhibitors and how do they affect a reaction?
Bind at another site on the enzyme and decreases the turnover number of an enzyme. This does not effect the affinity that an enzyme has for its substrate (Km) but prevents it from entering the active site - effectively lowering the concentration of effective enzyme. Affect Vmax but not Km
Why is Vmax not affected by competitive inhibition?
Adding enough substrate will always overcome the effects of the inhibitor and so have no effect on Vmax
Why is the Km affected by competitive inhibition?
Because the competitive inhibitor binds to the active site, it competes for the active site with the substrate. Therefore a higher concentration of substrate is needed to achieve half the maximum velocity - the Km is increased
