Enzyme Kinetics Practical Flashcards
Enzyme Kinetics
the study of the rates of an enzyme catalyzed reactions.
In enzyme kinetics,
Michaelis Menten - Low substrate concentration
At low substrate concentration, there is a steep increase in the rate of reaction with increasing substrate concentration.
The catalytic site is empty, waiting for the substrate to bind for much of the time.
The rate at which the product is formed is limited by the concentration of substrate.
The initial rate of an enzyme-catalyzed reaction
(V0)
Is directly proportional to the concentration of substrate [S]
In this region, the reaction is first order with respect to the substrate.
What happens when the concentration of substrate increases
The enzyme becomes saturated with substrate.
At high concentrations of substrate, there is a decreasing response as substrate concentration increases, until the maximum velocity (Vmax) is achieved at substrate saturation.
The reaction is now zero order with respect to the substrate
The rate of formation now depends on the activity of the enzyme itself, and adding more substrate will not affect the rate of reaction to any significant effect.
zero-order rate
a zero-order reaction has a rate that is independent of the concentration of the reactant(s). As such, increasing or decreasing the concentration of the reacting species will not speed up or slow down the reaction rate.
first order rate of reaction
A first-order reaction can be defined as a chemical reaction in which the reaction rate is linearly dependent on the concentration of only one reactant.
Draw the Michaelis Menten plot - the relationship between substrate concentration and initial velocity in an enzyme-catalyzed reaction
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What is Michaelis Menten
Relationship between Substrate
Concentration and Initial Velocity in an Enzyme-Catalysed Reaction
What is the Michaelis constant
Km , Km is the Michaelis-Menten constant which shows the concentration of the substrate when the reaction velocity is equal to one half of the maximal velocity for the reaction. It can also be thought of as a measure of how well a substrate complexes with a given enzyme, otherwise known as its binding affinity.
what is the Michaelis menten equation
V0 = Vmax[S]/ Km + [S]
Ks
The dissociation constant of the enzyme-substrate complex
1/Ks
The affinity constant of the enzyme for the substrate
Km and affinity
Km provides a measure of the affinity of an enzyme for the
substrate
A large value of Km indicates low affinity for substrate, a small Km indicates a high affinity for substrate
The Lineweaver Burk equation
When 1/ vo is plotted against 1/[S], a straight line is obtained, from which
the Michaelis constants Km and Vmax can be obtained as in Figure 2
created by plotting the inverse initial velocity (1/V0) as a function of the inverse of the substrate concentration (1/[S]). The Vmax can be accurately determined and thus KM can also be determined with accuracy because a straight line is formed.
Enzyme inhibition
Enzyme inhibitors are molecules that interact with an enzyme in some way and keep it from working in its usual manner
Competitive inhibitors
Competes with the substrate for binding at the enzyme active site
Competitive inhibitors
Competes with the substrate for binding at the enzyme active site
The binding of an inhibitor prevents substrate binding
A competitive inhibitor often has a similar chemical structure to the substrate, however, it is not acted on by the enzyme.
Thus, every time an inhibitor binds to the active site, the enzyme will no longer be able to catalyze the reaction and the rate will decrease.
enzyme-inhibitor complex
A competitive inhibitor reacts reversibly with the enzyme to form the enzyme-inhibitor complex. This is analogous to the enzyme-substrate complex.
Ki
The inhibitor constant. The dissociation of the enzyme- inhibitor complex.
What will a competitive inhibitor do to the Km of the enzyme.
Will increase the apparent Km of the enzyme for the substrate.
It causes it to require a higher substrate concentration to achieve Vmax.
Why is Vmax unchanged by a competitive inhibitor
Vmax can still be achieved if enough substrate is added to out-compete the inhibitor
A series of double-reciprocal plots , competitive inhibitor
A series of double reciprocal plots (Lineweaver Burke) plotted against each other at different concentrations, will all intersect at the same point.
1/Vmax on the Y-axis
Uncompetitive inhibition
Occurs when an inhibitor binds to the enzyme-substrate complex and in doing so renders the enzyme catalytically inactive.
Vmax and Km in uncompetitive inhibition
As some of the enzyme is rendered inactive, regardless of how much substrate is present Vmax is reduced, and hence in a double reciprocal plot 1/Vmax is increased.
The plot of 1/Vo against 1/[S] diff conc. uncompetitive inhibitor
A series of parallel lines,
the x-intercept shifts to the left
because km decreases and hence 1/km becomes larger
Noncompetitive inhibition
Occurs when the inhibitor binds at a site different from the active site.
The binding of the inhibitor at this site generally reduces the enzyme’s ability to convert substrates into products in the active site.
Vmax and Km - noncompetitive inhibitor
Vmax reduces as the enzyme can no longer function as well as it could in the absence of the inhibitor.
Vmax reduces, 1/Vmax will increase. The Km for the reaction will not change, because while the enzyme isn’t catalyzing the reaction well, it can still bind the substrate just as well as it did before.
Noncompetitive inhibition, Lineweaver burke
Plot of 1/Vo against 1/[S]
