Enzyme Activity And Kinetics Flashcards
Rate, velocity of enzyme catalyzed reaction
Measured at the beginning of the reaction when little amount of the product has formed ( initial velocity, Vo)
Enzyme activity
Enzyme activity is measured by determining the:
Amount of substrate consumed/ unit time
Amount of product formed/ unit time
Enumerate the Factors affecting enzyme activity
Substrate concentration
Temperature
pH
In substrate concentration, the rate of e-catalyzed reaction increases w/ substrate concentration until a ___ is reached
Maximal velocity (Vmax)
In substrate concentration, leveling off of the reaction rate at high substrate concentrations signifies that all active sites available for binding are already ____
Saturated with the substrate
Decrease in enzyme activity w/. Higher temp is due to what?
Due to temp-induced denaturation of enzyme
Increase in enzyme activity with temperature before a peak is reached is due to?
Due to more molecules having sufficient energy to pass over the energy barrier & form the products of the reaction
Effect of pH on the ionization of some functional groups in the active site
In order for a successful ES complex, some specific functional groups should be in ionized or unionized state
Decrease in enzyme activity during extremes of pH
Structure of the catalytically active protein is destabilized => denaturation
Study of rates of enzyme catalyzed reactions
Involves determination of kinetic parameters in order to describe the mode of action of enzymes
Consider relationships bet. Enzymatic activity & substrate concentration
Enzyme kinetics
Determining initial velocity (Vo) from the plots of product vs. time at different substrate concentration:
Determine the slope of the linear portion of the curve
Slope = initial velocity for a given substrate concentration
Plot initial velocity (Vo) vs. substrate concentration
Kinetic parameters
The fastest rate which the enzyme can catalyze the reaction
Vmax indicates that all available active site are occupied by the substrate (all involved in ES complex)
Maximum velocity (Vmax)
Refers to the substrate concentration that produces half maximal velocity
Michaelis constant (Km)
Smaller Km value in michael’s constant means:
Small amount of substrate is needed to obtain the ES complex
Enzyme has high affinity for the substrate
Math equation showing how reaction velocity of an enzyme reaction varies with substrate concentration & on Km
Michaelis - mentee equation
Vo= Vmax [s]
——————
Km + [s]
Michaelis-menten equation
case 1: [s]«< Km
Vo= Vmax [s] / Km
Vo is directly proportional to [s]
Michaelis-menten equation
Case 2 : [s]»_space;>Km
Vo= Vmax [s] / [s] => Vo=Vmax
When [s] exceeds km, rate is maximal
Michaelis-mentee equation
Case 3: [s] = Km
Vo= Vmax / 2
Rate is half maximal
Michaelis-mentee equation arranged into “double-reciprocal” where Vmax & Km can be determined
Lineweaver - Burke equation
1/Vo =1/Vmax + 1/ Vmax (1/[s])
y = b + m
An enzyme inhibition with a competitive w/ substrate and a non-competitive w/ substrate
Reversible inhibition
An enzyme inhibition that is covalently bind to enzyme to make it non functional
Irreversible inhibition
A reversible inhibition that can dissociate from the enzyme and diminishes rate of catalysis by reducing the proportion of enzyme molecules bound to a substrate.
Km value is increased - more substrate is needed to obtain the same reaction rate
High substrate concentration is needed
Vmax is unchanged
Competitive
A type of reversible inhibition that binds at sites other than the active site &does not compete with the substrate.
Vmax decreased, Km unchanged
Cannot be overcome by increasing substrate concentration
Non-competitive
Vmax decreased, Km decreased
Uncompetitive
Vmax decreased, Km increased
Mixed
