Enzyme Activity Flashcards
Define transition state
High energy intermediate that lies between substrate and product
Define activation energy
Minimum energy substrate must have to allow reaction
Name 2 ways to increase the rate of reaction
Temperature - increase number o molecules with activation energy
Concentration - increase chance of molecular collisions
Define enzymes
Biological catalysts that increase the rate of reaction by lowering the activation energy - facilitate formation of the transition state
List some important features of enzymes
Highly specific Unchanged after reaction Do not affect reaction equilibrium Increase rate of reaction Proteins May require associated cofactors
What is the clinical relevance of enzymes?
- Inheritable genetic disorders
- Overactive enzymes can cause disease
- Measurement of enzyme activity for diagnosis
- Inhibition of enzymes by drugs
Define active site
The active site of an enzyme is the place where substrates bind and where the chemical reaction occurs
How much of the enzyme does that active site occupy?
- The active site occupies a small part of the enzyme
Most enzymes are >100 aa but active site is only a few aa
Most of enzyme acts as a scaffold to create the active site
What is the active site formed from?
The active site is formed by amino acids from different parts of the primary sequence
Describe the shape of the active site and describe the 2 models of substrate fit
Active sites are clefts or crevices
Substrate molecules are bound in a cleft or crevice that usually excludes water
Active sites have a complementary shape to the substrate
“Lock and Key” hypothesis
Binding of substrates can induce changes in the conformation “Induced fit” hypothesis - The active site only forms a complementary shape after binding of the substrate
How are substrates bound to enzymes?
Substrates are bound to enzymes by multiple weak bonds
All types of non-covalent bonds
Binding must not be too tight!
What is V0?
Initial rate of reaction
What happened to enzyme activity as temperature increases?
Increases up to optimum
Then enzyme denatured
Activity decreases past optimum
Describe the shape of a graph of reaction velocity against substrate concentration
Rectangular hyperbola - it reaches a maximal velocity
What is the Michaelis-Menten model for enzyme catalysis?
The model proposes that a specific complex between the enzyme and the substrate is a necessary intermediate in catalysis.
V0 = Vmax[S]/Km+[S]
Predicts that a plot of V0 against [S] will be a rectangular hyperbola
NOT ALL ENZYMES OBEY THIS MODEL
Define Vmax
Maximal rate when all enzyme active sites are saturated with substrate
Define Km
Substrate concentration that gives half maximal velocity
Km = Michaelis constant (M)
M = moles/litre
What is the significance of Km values?
Km values give a measure of the affinity of an enzyme for it’s substrate
Low Km = high affinity for substrate
High Km = low affinity for substrate
What is the significance of Vmax/V0 values?
Vmax/V0 values are rates
Measured in amounts per unit time
Often expressed as a standardised rate: per litre (L) of serum or per gram (g) of tissue
The rate of an enzyme catalysed reaction is proportional to the concentration of enzyme
Double the amount of enzyme – double the rate BUT – not the standardised rate
What is 1 unit?
1 unit = the amount of enzyme that converts 1mmol of product per min under standard conditions
What is the Lineweaver-Burk plot
Michaelis-Menten equation can be rearranged to give a linear plot
V0 = Vmax[S]/Km+[S]
Y axis = 1/V X axis = 1/[S] Gradient = Km/Vmax Y intercept = 1/Vmax X intercept = -1/Km
Allows for easy estimation of Km and Vmax
What are enzyme inhibitors?
Molecules that slow down or prevent an enzyme reaction
- including many drugs
What are the types of enzyme inhibitors?
- Irreversible: Bind very tightly, generally form
covalent bond(s)
Example: nerve gases, such as sarin - Reversible: Non-covalent; can freely dissociate.
i) Competitive – binds at active site.
ii) Non-competitive – binds at another site on the enzyme - the allosteric site
Describe competitive inhibition
The competitive inhibitor resembles the substrate and binds to the active site, reducing the proportion of enzyme molecules bound to the substrate.
How does competitive inhibition affect Km and Vmax?
Adding enough substrate will always overcome the effect of the inhibitor, so no effect on Vmax
Because the inhibitor competes with the substrate for the active site Km increases
Slower speed to reach same Vmax
Describe non-competitive inhibition
Non-competitive inhibitor binds at an alternative site (allosteric site) and decreases the turnover number of the enzyme
How does non-competitive inhibition affect Km and Vmax?
Lowers Vmax but Km unaffected
Same speed reaction but Vmax is lower
