Chapter 7: Enzyme Kinetics and Inhibition Flashcards
What is enzyme kinetics?
It’s the use of math to fully describe enzyme activity by quantifying an enzyme’s:
* catalytic power
* substrate affinity
* its response to inhibitors
How can the rate of a reaction be monitored?
Velocity (rate of reaction): concentration vs time
What is the relationship between reaction velocity and enzyme concentration?
(v) = either the rate of disappearance of the substrate (S) or the rate of appearance of the product (P). The reaction is faster if there is a catalyst present
What are the parameters between the hyperbolic curve?
When the enzyme concentration is held constant, the reaction velocity varies with the substrate concentration, but in a nonlinear fashion
What are the two reactions described by the Michaelis-Menten equation?
- E and S collide to form ES (bimolecular reaction)
- ES goes back to E and S or forms P and E (both uni-molecular reactions)
What is the rate equation for the first-order reactions?
In a unimolecular or first-order reaction, the velocity is dependent on the concentration of only one substrate
What is the steady-state assumption and its implications?
[ES] has a constant value. The concentration of ES remains constant until nearly all the substrate has been converted to product.
What is the rate-limiting step of the Michaelis-Menten reactions?
k2
What are the initial velocities assumption and its implications?
Initial velocity assumption! k-2 can be eliminated
What is the Michaelis-Menten equation and the meaning of its terms?
It is the rate equation for an enzyme-catalyzed reaction and is the mathematical description of the hyperbolic curve
How to interpret the Michaelis-Menten plot
Enzyme inhibitors.
Lower the Km the higher the affinity
Higher the km the lower the affinity
What is the definition and most common interpretation of the Michaelis constant, KM?
- Since KM is the substrate concentration at which the
reaction velocity is half-maximal, it indicates how efficiently an en-
zyme selects its substrate and converts it to the product. - Is the [S] at which velocity is
half-maximal (Vmax/2) - It’s often used as a measure of an enzyme’s affinity for a substrate
- The lower the KM, the higher the affinity
What is the meaning of turnover number or catalytic constant, kcat?
kcat is the rate constant of the reaction when the enzyme is saturated with substrate.
* It indicates how fast an enzyme can
act after it has bound its substrate
* It’s the rate constant of the reaction
when the enzyme is saturated with substrate
kcat is also known as the enzyme’s turnover number because it is the number of catalytic cycles that each active site undergoes per unit time, or the number of substrate molecules transformed to product molecules by a single enzyme in a given period of time.
How can the catalytic efficiency be obtained?
The quantity kcat/KM satisfies this requirement.
An enzyme’s effectiveness as a catalyst depends on how avidly it binds its substrates
and how rapidly it converts them to products. Thus, a measure of catalytic efficiency must reflect both binding and catalytic events.
The value of kcat/KM, more than either KM or kcat alone, represents the enzyme’s overall ability to convert substrate to product.
What is the Lineweaver-Burk plot and how are the Vmax and KM can be obtained from the plot?
A linear representation of M-M kinetics data
What types of enzymes that do not fit the Michaelis-Menten model kinetics?
- Multiple substrates and products: example: transketolase)
- Multiple-step Reactions or ping pong reactions (example: transketolase)
- Allosteric enzymes (nonhyperbolic): oligomeric enzymes with multiple active sites, do not obey the Michaelis–Menten rate equation and therefore do not yield hyperbolic velocity versus substrate curves (ex. Cooperative behavior that occurs in hemoglobin, when O2 binding to the heme group in one subunit alters the O2 affinity of the other subunits)
What are the characteristics of allosteric enzymes?
- The result of allosteric behavior is a sigmoidal
- cooperative behavior: a substrate at one active site can affect the catalytic activity of the other active sites.
- Multimeric enzymes
- non-hyperbolic curve
What is the meaning of cooperative behavior?
Cooperative behavior occurs when the enzyme subunits are structurally linked to each other so that a substrate-induced conformational change in one subunit elicits conformational changes in the remaining subunits.
(Cooperative behavior also occurs in hemoglobin, when O2 binding to the heme group in one subunit alters the O2 affinity of the other subunits)
What is the general interpretation of a sigmoidal plot?
The velocity versus substrate curve is sigmoidal rather than hyperbolic when substrate binding to one active site in an oligomeric enzyme alters the catalytic activity of the other active sites. The maximum reaction velocity is Vmax, and K M is the substrate concentration when the velocity is halfmaximal.
What are the different types of enzyme inhibitors?
- Irreversible
* Inhibitor forms a covalent bond: Any reagent that covalently modifies an amino acid side chain in a protein can potentially act as an irreversible enzyme inhibitor.
* Some inhibitors are called
suicide inhibitors because they enter the enzyme’s active site and begin to react, just as a normal substrate would. However, they are unable to undergo the complete reaction and hence become “stuck” in the active site. - Reversible
* Competitive inhibition (ubiquitous): In this situation, the inhibitor is a substance that directly competes with a substrate for binding
to the enzyme’s active site
* Noncompetitive inhibition: bind at opposite end of active site
* Uncompetitive inhibition
Why can aspirin inhibit COX?
Inhibition of COX-1 and COX-2 activity by aspirin is attributed to the covalent modification of active site serine residues. Acetylation of these side-chain hydroxyl groups results in irreversible inhibition through steric blockade of the active site.
5-fluorouracil can inhibit thymidylate synthase
Some irreversible enzyme inhibitors are called suicide substrates because they enter the enzyme’s active site and begin to react, just as a normal substrate would. However, they are unable to undergo the complete reaction and hence become “stuck” in the active site. For example, thymidylate synthase is the enzyme that converts the nucleotide de- oxyuridylate (dUMP) to deoxythymidylate (dTMP) by adding a methyl group to C5.
What are the characteristics of competitive inhibitors and effect on Vmax and KM?
A competitive inhibitor increases the apparent KM of the enzyme but
does not affect kcat or Vmax.
What is the Lineweaver-Burk plot for competitive inhibition?
