Enzyme Activity - Kinetics And Inhibition Flashcards
Chemical reactions - what is transition and activation state
Transition state = high energy intermediate that lies between S and P (substrate and product)
Activation energy = minimum energy S must have to allow reaction
Increasing the rate of a reaction
1) Temperature - Increase no. of molecules with activation energy
2) Concentration - Increases chance of molecular
collisions
ENZYMES - Biological catalysts that increase the rate of reaction by lowering the activation energy
Facilitate formation of the transition state
Important features of enzymes
Highly specific
Unchanged after the reaction
Do not affect the reaction equilibrium
Increase the rate of reaction
They are Proteins
May require associated cofactors
Defects can lead to Inheritable genetic disorders
Overactive enzymes can cause disease
Measurement of enzyme activity for diagnosis
Inhibition of enzymes by drugs
Active sites
The active site of an enzyme is the place where substrates bind and where the chemical reaction occurs
- 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 - The active site is formed by amino acids from different parts of the primary sequence - on the AA chain the parts of active site are far apart - but are physiological close due to protein folding
- 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
The active site of the enzyme is complementary in shape to that of the substrate
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
- Substrates are bound to enzymes by multiple weak bonds
All types of non-covalent bonds
Binding must not be too tight!
Enzyme kinetics
Can plot velocity of enzyme Kinectics when looking at product production over time
Vo is the initial rate of the reaction
Rate of reaction can vary depending on temperature and pH the enzyme works best at e.g. pepsin works best at a pH of 2
Reaction rate as a function of substrate concentration - An enzyme-catalysed reaction reaches a maximum velocity: the plot often has the shape of a rectangular hyperbola.
Michaelis-Menten equation - predicts that a plot of Vo versus substrate concentration will be a rectangular hyperbola. (Not all enzymes obey this model)
Vmax = maximal rate when all enzyme active sites are saturated with substrate Km = substrate concentration that gives half maximal velocity
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
Hexokinase Km 0.1mM
Glucokinase Km 5mM
HK is always active whereas GK only becomes active when glucose levels peak after feeding - HK always working, GK only recruited after feeding to help deal with increased substrate
The significance of Vmax/Vo values
Vmax / Vo values are rates
Measured in amounts per unit time
1 unit = the amount of enzyme that converts 1mmol of product per min under standard conditions
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
The line weaver-Burk plot
The Michaelis-Menten equation can be rearranged to give a linear plot
Allows for easy estimation of Km and Vmax from linear plot
Further to the left the X- intercept is higher the affinity the enzyme has
Enzyme inhibitors
Molecules that slow down or prevent an enzyme reaction - including many drugs
- 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. Affects Km, not Vmax
(ii) Non-competitive – binds at another site on the enzyme, Affects V max but not Km
Competitive inhibition
The competitive inhibitor resembles the substrate and binds to the active site, reducing the proportion of enzyme molecules bound to the substrate - can be overcome if you overload substrate concentration as it outcompetes inhibitor
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
Competitive inhibition = Km increases, Vmax unaffected
Non-competitive inhibition
Non-competitive inhibitor binds at an alternative site and decreases the turnover number of the enzyme, thus lowering Vmax
Compounds binding outside the active site can also activate rather than inhibit
As the enzyme active site is changed, then it cant be saturated with the substrate, therefore Vmax will decrease, but the rate at which the enzymes (that do work) will not change, hence Km will stay the same
Non-competitive inhibition - Km unaffected, Vmax decreases
