Enzyme models, environmental effect, velocity, substrate level, Michaelis Menten and its derivatives Flashcards
Enzyme definition and function
Organic catalyst that speeds up chemical reactions in the cell, without themselves being changed.
Enzyme catalyzed reactions are 10^3 to 10^17 times faster than uncatalyzed reactions. Without enzymes, reactions in the cell would be extremely slow.
3 characteristics of enzyme function
Enzymes are specific
Enzymes remains unchanged after they are used as catalyst
Enzymes work in either directions in reversible, direction depends on the amount of substrate/product present and the reaction continues until equilibrium is reached
Holoenzymes and Apoenzymes
Holoenzymes are the active form of enzymes, they represent the apoenzyme bound to its necessary cofactors or prosthetics groups.
Apoenzymes are enzymes that require a cofactor but are not bound by one.
Enzyme cofactors function
The 3 types of enzyme cofactors
Present at the active site of the enzyme
Non-protein
1. Activators can be metal ions or other inorganic ions
Examples Zn in metalloproteases
2. Coenzymes Organic non-protein compounds Many which are derived from vitamins Not covalently bound Examples is Co-enzyme A used to form acetyl-COA in kreb cycle
3.Prosthetics groups
Metal ion or an organic compound that is covalently bound to an enzyme
Example is Heme in hemoglobin
Enzyme active site
Groove in the enzyme that binds to specific substrates, a small part of the enzyme
Specific shape
Only specific substrate molecules will fit into the active site to form an enzyme-substrate complex
Catalytically active amino acids within the active site of the enzyme site of the enzyme act on the substrate t convert it to product
How does an enzyme work
An enzyme works by catalyzing reactions that convert the substrates to products via a temporary enzyme-substrate complex
What are the 3 ways which enzymes work
- Activation energy
- Lock and Key model
- Induced fit model
Activation energy and the 2 ways which enzymes lower activation energy
Activation energy is the initial energy needed to start a reaction between substrate molecules
Enzymes lower the activation energy by bringing the substrates closer together, which increase the chance of successful reactions
They distort and weakens bonds between the substrates?? (ask)
What is a lock and key model
- Describe that the shapes of the substrate and the active site of enzymes are fixed
- Only a specific substrate can fit into the active site
What is a induced fit model
- Describes that the shape of the substrate and active site are not fixed
- Binding of substrate induce a conformational change in the active site of enzyme
List 4 factors that affect the rate of reaction
Enzymes and Substrate concentration
Heat and pH
Presence of Cofactors - Metal ions, organic and organometallic species
Presence of Inhibitors
Describe how enzymes concentration affects the rate of reaction
At low enzyme concentration, there is great competition for the active sites and the rate of reaction is low.
As the enzyme concentration increases, there are more active sites and the reaction can proceed at a faster rate.
Eventually, increasing the enzyme concentration beyond a certain point has no effect because the substrate concentration becomes the limiting factor.
Describe how substrate concentration affects the rate of reaction
At a low substrate concentration there are many active sites that are not occupied. This means that the reaction rate is low.
When more substrate molecules are added, more enzyme-substrate complexes can be formed. As there are more active sites, and the rate of reaction increases.
Eventually, increasing the substrate concentration yet further will have no effect.
The active sites will be saturated so no more enzyme-substrate complexes can be formed.
Describe the enzyme reaction graph
Initial velocity - The reaction rate of an enzymatic reaction is always fastest at the beginning of the reaction when there is the greatest concentration of substrate.
The graph represents the amount of product formed. At first, the amount of product formed increases, then the rate slows down as the concentration of substrate decreases. The fastest rate of product formation is at the beginning and is called the initial velocity. It is more likely to get an effective collision between enzyme and substrate molecules if there are more substrate molecules present; as in the beginning of the reaction. If the enzyme is kept constant and there is an increase in the amount of substrate, there will be an increase in the initial velocity until a saturation point is reached.
Rates increase until a saturated point, max velocity also known as Vmax
Faster reaction but it reaches a saturation point when all the enzyme molecules are occupied.
If you alter the concentration of the enzyme then Vmax will change too
Saturation point is the point when there are enough substrate molecules to completely saturate the enzyme’s active sites, Vmax is the rate under these conditions.
Effect of pH on enzyme reaction rate
Optimum pH where enzymes work their best
If the pH changes much from the optimum, the chemical nature of amino acids can change.
H+ ions can affect the bonding between R groups and secondary structures, so the tertiary structure of the enzymes can break down
The active site will be disrupted and the enzyme will be denatured
Denaturation
Shape of active site is distorted and substrate molecule will no longer fit in it
Change in ionization will affect the binding of substrate with the active site
At pH values slightly different from enzyme optimum value, small changes in the charges of enzymes and its substrate molecule will occur
Effect of pH
pH can also affect the reaction rates.
Most enzymes work best at a range of 6 to 8, but there are some exceptions, such as pepsin.
If the environment changes much from the optimum pH, bonds at the active site are affected, denaturing the enzyme - Changes the ionization state of amino acid side chains at the active site
Bell shaped curve because both an increase and a decrease in pH from the optimum can denature the enzyme.
As the pH moves further from the optimum pH, it increases the likelihood that the active site is disrupted.
Effect of temperature
Enzymes work best at an optimum temperature.
Below this, an increase in temperature provides more kinetic energy to the molecules involved. The numbers of collisions between enzyme and substrate will increase so the rate will too.
Above the optimum temperature, and the enzymes are denatured. Bonds holding the structure together will be broken and the active site loses its shape and will no longer work
Heat energy cause more collisions between enzymes and substrates, which lead to temperature increasing products formed.
Optimum temperature for humans is 37 degree Celsius where products formed are the highest rate.
Enzymes denature at high temperature beyond optimum temperature so rate falls rapidly
Conjugated enzymes vs simple enzymes
Conjugated enzymes are simple enzymes that have other chemical groups attached to it.
Enzyme kinetics
Study the rate of enzyme catalyzed reactions using Michaelis mentis kinetics, its derivatives and inhibition kinetics
Velocity definition
Velocity - Rate of reaction - rate of S disappearing over time or appearance of P overtime
Initial velocity definition and 4 characteristics of initial velocity
Measured at time zero > rate of reaction is fastest
Maximum amount of substrate is available
No reverse reaction
No feedback inhibition
No loss in enzyme activity
Plot of reaction velocity vs substrate concentration
Varying amounts of substrate are added to a fixed amount of enzyme. The reaction velocity is measured for each substrate concentration and plotted. The resulting curve takes the form of a hyperbola (a mathematical function in which the values initially increase steeply but eventually approach a maximum level).
Understanding the Michaelis menten curve
Plotting V as a function of [S],
At low values of [S], the initial velocity,V0, rises almost linearly with increasing [S].
But as [S] increases, the gains in V0 level off
Represents the maximum velocity of the reaction, designated Vmax
The substrate concentration that produces a V0 that is one-half of Vmax is designated the Michaelis-Menten constant, Km
Km is (roughly) an inverse measure of the affinity or strength of binding between the enzyme and its substrate. The lower the Km, the greater the affinity (so the lower the concentration of substrate needed to achieve a given rate).
How to find Km from Vmax
Half Vmax = Vmax/2, at this Vmax/2, [S] is equal to Km.
Michaelis constant 3 extra things to note
It is independent of [E], enzyme concentration
It measures “relative affinity” of an enzyme for its substrate
If there is more than 1 substrate, then each substrate has its own Km
Understanding Michaelis constant, Km
The concentration of substrate [S] required to make the reaction go at half its maximum velocity
Km = substrate concentration at ½ Vmax
Measure of affinity that the enzyme has for a substrate
A low Michaelis constant means that there is a high affinity between the enzyme and substrate
A high Michaelis constant means that there is a low affinity between the enzyme and substrate
Vmax
Vmax is the theoretical maximal rate of the reaction - but it is NEVER achieved in reality
To reach Vmax would require that ALL enzyme molecules are tightly bound with substrate
What is the formula of michaelis menten
Vo = Vmax.[S] / (KM + [S])
Kcat
Kcat - number of substrate molecules converted into product per unit time at a single catalytic site
Kcat - measured at optimum pH and temperature.
Kcat = Vmax / {E}t
Michaelis menten plot
Reaction velocity on y axis and Substrate concentration on x axis
However, it will not give accurate values of Km as Vmax is never reached as a hyperbola
Lineweaver Burk plot
y = mx+c
1/Vo = Km/Vmax x 1/ {S} + 1/Vmax
Y axis is 1/{Vo}
X axis is 1/{S}
A double reciprocal graph
The x intercept is -1/Km
The y intercept is 1/Vmax
Advantage and Disadvantage of Lineweaver Burk plot
Advantage : Lineweaver-Burk equation or double reciprocal plot is useful graphical
Disadvantage : y-axis takes the reciprocal of the rate of reaction in turn increasing any small errors in measurement.
At high substrate concentration, data points crowded to one side.
The least accurately measured points (that is high[S] are given the highest weightage)
y-axis takes the reciprocal of the rate of reaction in turn increasing any small errors in measurement.
Eadie Hofstee plot
Y = mx + c
y = Vo
x = Vo/{S)
c = y-intercept
m = -Km
y axis is Vo or velocity
x axis is Vo/{S)
slope is a negative Km
y-intercept is the Vmax
Advantage and Disadvantage of Eadie Hofstee plot
Advantage: Equal weight of data points and able to identify outliers
Disadvantage: V is represented in both axis. Experimentally, this quantity is generally subject to more error than substrate concentration
Prosthetic groups
Cofactors that are tightly bound, that can be metal ions or organic compounds.
Coenzyme
Coenzymes are organic compounds that can be tightly and loosely bound.
